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수의학 박사 학위논문
Genomic research of bacteriophage and
bacteriophage therapy against Aeromonas
hydrophila and Vibrio parahaemolyticus
박테리 파아지 유전체연구 및 Aeromonas hydrophila
Vibrio parahaemolyticus에 대한 박테리 파아지 치료법
2014 년 2 월
서울대학교 대학원
수의학과 수의공중보건학 전공
전 진 우
A Dissertation for the Degree of Doctor of Philosophy
Genomic research of bacteriophage and
bacteriophage therapy against Aeromonas
hydrophila and Vibrio parahaemolyticus
By
Jin Woo Jun
February, 2014
Major in Veterinary Public Health
Department of Veterinary Medicine
The Graduate school of Seoul National University
Genomic research of bacteriophage and
bacteriophage therapy against Aeromonas
hydrophila and Vibrio parahaemolyticus
By
Jin Woo Jun
Supervisor: Professor Se Chang Park, D.V.M., Ph.D.
A dissertation submitted to the faculty of the Graduate School of Seoul
National University in partial fulfillment of the requirements for the degree of
Doctor of Philosophy in Veterinary Public Health
February, 2014
Department of Veterinary Public Health
College of Veterinary Medicine
The Graduate school of Seoul National University
Genomic research of bacteriophage and
bacteriophage therapy against Aeromonas
hydrophila and Vibrio parahaemolyticus
박테리 파아지 유전체연구 및 Aeromonas hydrophila Vibrio
parahaemolyticus에 대한 박테리 파아지 치료법
지도교수: 박 세 창
이 논문을 수의학박사 학위논문으로 제출함
2013 년 11 월
서울대학교 대학원
수의학과 수의공중보건학 전공
전 진 우
전진우의 박사학위 논문을 인준함
2013 년 12 월
위 원 장 이 병 천 (인)
부위원장 박 세 창 (인)
위 원 조 성 준 (인)
위 원 신 기 욱 (인)
위 원 Mahanama De Zoysa (인)
i
Abstract
Genomic research of bacteriophage and
bacteriophage therapy against Aeromonas
hydrophila and Vibrio parahaemolyticus
Jin Woo Jun
Department of Veterinary Public Health
College of Veterinary Medicine
The Graduate School of Seoul National University
Aeromonas spp. are primary organisms of normal aquatic microflora, and recently, there
has been an increasing appreciation of their role as waterborne pathogens of fish and
humans. Aeromonas hydrophila is a motile aeromonad that can cause disease in fish,
resulting in high rates of mortality wherever outbreaks occur. There has been an increasing
incidence of antimicrobial resistance among Aeromonas sp. isolated from aquaculture
environments. Multiple-antibiotic-resistant A. hydrophila exists in aquaculture systems and
contributes to the high rate of mortality within the fish industry in Korea. Although the
majority of the loach population in Korea is cultured and A. hydrophila is one of the main
causes of mass mortality in these fish, no effective method has been proposed for the
control of A. hydrophila infection in aquaculture, except for the application of additional
antibiotics. To investigate methods to control the mass mortality of cyprinid loaches
(Misgurnus anguillicaudatus) caused by multiple-antibiotic-resistant Aeromonas
hydrophila on a private fish farm in Korea, bacteriophages (phages), designated pAh1-C
ii
and pAh6-C, were isolated from the Han River in Seoul. The two isolated phages were
morphologically classified as Myoviridae and showed similar infection patterns for A.
hydrophila isolates. The two phages possessed approximately 55 kb (pAh1-C) and 58 kb
(pAh6-C) of double-stranded genomic DNA, and their gDNAs showed different restriction
endonuclease digestion patterns. Both phages showed efficient bacteriolytic activity
against fish-pathogenic A. hydrophila from loaches. The latent periods of the phages were
estimated to be approximately 30 min (pAh1-C) and 20 min (pAh6-C), while the burst
sizes were 60 PFU/cell (pAh1-C) and 10 PFU/cell (pAh6-C). The phages proved to be
efficient in the inhibition of bacterial growth, as demonstrated by their in vitro bactericidal
effects. Additionally, a single administration of either phage to cyprinid loaches resulted in
noticeable protective effects, with increased survival rates against A. hydrophila infection.
These results suggest that the phages pAh1-C and pAh6-C constitute potential therapeutic
agents for the treatment of A. hydrophila infection in fish.
Vibrio parahaemolyticus is one of the most important causes of gastroenteritis. It is
associated exclusively with the consumption of raw or improperly cooked contaminated
seafood, especially oysters. V. parahaemolyticus is recognized as an important human
pathogen globally. East Asians, especially Koreans and Japanese consume a unique diet.
Koreans enjoy a wide variety of both raw finfish and shellfish. Although raw oysters have
such high densities of V. parahaemolyticus that the consumption of raw oysters is known to
cause illness in humans, almost all Koreans prefer raw oysters to already cooked oysters
because of their fresh taste and high nutritional value. V. parahaemolyticus pandemic
strains, such as O3:K6, are responsible for the current pandemics in many countries.
Emergence of Vibrio species that are resistant to multiple antibiotics has been recognized
as a serious global clinical problem. Recently isolated V. parahaemolyticus pandemic
iii
strains have displayed multiple antibiotic resistance, increasing concerns about possible
treatment failure. Alternatives to conventional antibiotics are needed, especially for the
multiple-antibiotic-resistant V. parahaemolyticus pandemic strain. A bacteriophage,
designated pVp-1, that was lytic for V. parahaemolyticus was isolated from the coast of the
Yellow Sea in Korea. The phage showed effective infectivity for multiple-antibiotic-
resistant V. parahaemolyticus and V. vulnificus, including V. parahaemolyticus pandemic
strains. The therapeutic potential of the phage was studied in a mouse model of
experimental infection using a multiple-antibiotic-resistant V. parahaemolyticus pandemic
strain. Phage-treated mice displayed protection from a V. parahaemolyticus infection and
survived lethal oral and intraperitoneal bacterial challenges. This is the first report, to the
best of the knowledge, of phage therapy in a mouse model against a multiple-antibiotic-
resistant V. parahaemolyticus pandemic strain infection.
The complete genome sequence of a novel marine siphovirus pVp-1, which was
isolated from the coastal water of the Yellow sea in Korea and infects V. parahaemolyticus,
was reported. The double-stranded DNA genome of pVp-1 is composed of 111 kb with a G
+ C content of 39.71%. The genome encodes a total of 157 ORFs. Genome sequence
analysis of pVp-1 and comparative analysis with the homologous phage T5 revealed that
there is a degree of similarity between pVp-1 and T5, thus indicating a close genetic
relatedness between pVp-1 and T5. Genomic comparison of pVp-1 with the phage T5
revealed that these two phages are highly similar in gene inventory.
Based on these results, it can be concluded that Aeromonas phages that infect antibiotic-
resistant A. hydrophila strains could be considered as alternative therapeutic or
prophylactic candidtates against Aeromonas infections in aquaculture. In addition, phage
treatment trials in the mouse model for V. parahaemolyticus CRS 09-17 demonstrated that
iv
the application of pVp-1 can protect from a V. parahaemolyticus infection, and pVp-1 can
be used as a therapeutic agent to reduce the impact of epidemics caused by multiple-
antibiotic-resistant pandemic strains.
Key words: Aeromonas hydrophila, Bacteriophage (phage), Vibrio parahaemolyticus,
Multiple-antibiotic-resistant pandemic strains, Therapeutic agent.
Student number: 2011-31100
v
Contents
Abstract ............................................................................................................... i
Contents ............................................................................................................. v
List of figures .................................................................................................. viii
List of tables ...................................................................................................... ix
Abbreviations ..................................................................................................... x
General Introduction ......................................................................................... 1
Literature Review
A. Aeromonas hydrophila................................................................................... 7
A.1. Perspective of Aeromonadaceae ................................................................. 7
A.2. Aeromonadaceae species ............................................................................ 8
A.3. Aeromonas and ecosystems ........................................................................ 9
A.4. Virulence factors of Aeromonas ................................................................ 10
A.5. Aeromonas and aquatic environments ....................................................... 11
A.6. Aeromonas and fish diseases ..................................................................... 12
A.7. Antimicrobial resistance ........................................................................... 12
B. Vibrio parahaemolyticus .............................................................................. 14
B.1. Perspective of Vibrio ................................................................................ 14
B.2. Viewpoint of Vibrio parahaemolyticus ..................................................... 15
B.3. Occurrence of Vibrio parahaemolyticus .................................................... 16
B.4. Incidence of V. parahaemolyticus food poisoning ..................................... 17
B.5. Virulence factors ...................................................................................... 19
B.6. Pandemic strains of V. parahaemolyticus .................................................. 19
C. Bacteriophage (phage) ................................................................................ 21
C.1. General description ................................................................................... 21
vi
C.2. Phage application in aquaculture ............................................................... 23
C.3. Phage therapy ........................................................................................... 24
D. References ................................................................................................... 25
Chapter I
Protective effects of the Aeromonas phages pAh1-C and pAh6-C against mass
mortality of the cyprinid loach (Misgurnus anguillicaudatus) caused by
Aeromonas hydrophila
Abstract ............................................................................................................. 37
1.1. Introduction .............................................................................................. 39
1.2. Materials and Methods.............................................................................. 40
1.3. Results ...................................................................................................... 45
1.4. Discussion ................................................................................................ 49
1.5. References ................................................................................................ 52
Chapter II
Bacteriophage Therapy of a Vibrio parahaemolyticus Infection Caused by a
Multiple Antibiotic Resistant O3:K6 Pandemic Clinical Strain
Abstract ............................................................................................................. 66
2.1. Introduction .............................................................................................. 67
2.2. Materials and methods .............................................................................. 68
2.3. Results ...................................................................................................... 72
2.4. Discussion ................................................................................................ 75
2.5. References ................................................................................................ 78
Chapter III
Complete genome sequence of a novel marine siphovirus pVp-1, infecting
Vibrio parahaemolyticus
Abstract ............................................................................................................. 92
vii
3.1. Introduction .............................................................................................. 93
3.2. Materials and Methods.............................................................................. 93
3.3. Results ...................................................................................................... 94
3.4. Discussion ................................................................................................ 95
3.5. References ................................................................................................ 96
General conclusion......................................................................................... 110
Abstracts in Korean ....................................................................................... 112
List of published articles ................................................................................ 116
List of conference attendances ...................................................................... 125
Acknowledgements ........................................................................................ 135
viii
List of Figures
Chapter I
Figure 1.1. Phage plaques formed in double layer agar plates with the indicator host strain, A.
hydrophila JUNAH, and electron micrographs of phages.
Figure 1.2. One-step growth curve of pAh1-C and pAh6-C.
Figure 1.3. Stability of pAh1-C and pAh6-C in the presence of various organic solvents, pHs, and
temperatures.
Figure 1.4. Bactericidal effects of pAh1-C and pAh6-C against A. hydrophila JUNAH.
Figure 1.5. Cumulative mortality of different treatment groups following challenge with A.
hydrophila via IP injection.
Chapter II
Figure 2.1. Electron micrograph of negatively stained phage pVp-1.
Figure 2.2. One-step growth curve of pVp-1.
Figure 2.3. The bacteriolytic effect of pVp-1 against V. parahaemolyticus CRS 09-17.
Figure 2.4. Experimental infection of mouse model.
Figure 2.5. Kinetics of pVp-1 in the mouse model.
Figure 2.6. Effect of phage treatment on V. parahaemolyticus infection in mice.
Figure 2.7. CFU and PFU values of the stomach/intestine.
Figure 2.8. Histopathologic features of the intestines of mice infected with V. parahaemolyticus
CRS 09-17 and treated with the phage pVp-1.
Figure 2.9. The sensitivity of pVp-1 to various organic solvents, pH, temperatures, and exposure
to UV light.
Figure 2.10. Antibody titers in mice (n = 5) to repeated injections of phage pVp-1.
Chapter III
Figure 3.1. Genome map of phage pVp-1.
Figure 3.2. Genome comparison of phage pVp-1 to its relative phage (T5) using Artemis
Comparison Tool (ACT).
ix
List of Tables
Literature review
Table I. Companies involved in phage application.
Chapter I
Table 1.1. Bacterial strains used in this study and infectivity of phage pAh1-C and pAh6-C.
Table 1.2. Pathogenicities of phage-sensitive parent cells and phage-resistant variants of A.
hydrophila (JUNAH) for loach.
Table 1.3. SDS-PAGE profiles of pAh1-C and pAh6-C virion and their protein profiles by liquid
chromatography-mass spectrometry (LC-MS/MS) analysis.
Chapter III
Table 3.1. Predicted genes and gene products of pVp-1.
x
Abbreviations
CFU Colony Forming Unit
EOP Efficiency Of Plating
FDA Food and Drug Administration
IP Intra Peritoneally
LC-MS/MS Liquid Chromatography-tandem Mass Spectrometry
MOI Multiplicity Of Infection
OD Optical Density
ORF Open Reading Frame
pAh Phage against Aeromonas Hydrophila
PFU Plaque Forming Unit
pVp Phage against Vibrio Parahaemolyticus
SDS-PAGE Sodium Dodecyl Sulfate-Poly Acrylamide Gel Electrophoresis
SPSS Statistical Package for the Social Sciences
TEM Transmission Electron Microscopy
TSA Tryptic Soy Agar
TSB Tryptic Soy Broth
1
General introduction
The genus Aeromonas is a member of the family Aeromonadaceae that are primarily
aquatic organisms found in water. Some Aeromonas sp. are pathogenic for humans as well
as fish (27). The organisms in this family produce a clear zone of β-hemolysis on blood
agar (11). A. hydrophila is a member of motile aeromonads and it can cause disease in fish,
resulting in high mortality (1, 2, 5, 6, 18). There has been an increasing incidence of
antimicrobial resistance among Aeromonas sp. isolated from aquaculture environments (24,
25, 26). Five classes of genetically distinguishable tetracycline resistance determinants,
designated A through E, have been described among aerobic enteric gram-negative bacteria
(20). Several studies have shown tetE to be the predominant determinant among the
different classes of tetracycline-resistant genes (4, 19, 25).
The loach (Misgurnus spp.) is a member of the Cobitidae family (Lacepede, 1803) and
inhabits freshwater systems by nature (13). Two species of loaches (Misgurnus spp.), the
mud loach (M. mizolepis) and the cyprinid loach (M. anguillicaudatus), are cultured mostly
for food and sometimes for Buddhism ceremonies in Korea (12). The annual demand for
loaches in Korea and Japan was over 100,000t in 2004 due to its high nutritional value and
use in folk medicine (8). Aquaculture of loach in 2008 was over 432t in Korea (14).
Jeollabuk-do province in Korea is famous for the aquaculture of loaches, with over 384t in
2008, which was 89% of the total loach aquaculture in the entire country (7).
There have been few reports about A. hydrophila in Korea since the previous
publication about isolation of A. hydrophila from rainbow trouts in Korea (16). Although
the majority of the loach population is cultured and A. hydrophila is one of the main causes
of its mass mortality in Korea, there was only little knowledge of this bacterium from
2
cyprinid loach.
Vibrio parahaemolyticus is one of the most important causes of gastroenteritis (3). It is
associated exclusively with the consumption of raw or improperly cooked contaminated
seafood, especially oysters (3). V. parahaemolyticus is recognized as an important human
pathogen globally (3, 9, 22). According to the official statistics issued by the Korea Food
and Drug Administration (10), V. parahaemolyticus is one of the most common causes of
food-borne disease in Korea, and caused 17 outbreaks with a total of 663 patients in 2005,
25 outbreaks (547 patients) in 2006, 33 outbreaks (634 patients) in 2007, 24 outbreaks
(329 patients) in 2008, and 12 outbreaks (106 patients) in 2009.
East Asians, especially Koreans and Japanese consume a unique diet. Koreans enjoy a
wide variety of both raw finfish and shellfish. Although raw oysters have such high
densities of V. parahaemolyticus that the consumption of raw oysters is known to cause
illness in humans (3), almost all Koreans prefer raw oysters to already cooked oysters
because of their fresh taste and high nutritional value. Also, seafood cross-contaminated
with raw oyster can cause high risk for V. parahaemolyticus infections in the United States
(3). In Korea especially, all seafood in fishery markets, including oysters, is sold in the
same seawater and tanks, causing cross-contamination.
Virulence of V. parahaemolyticus is commonly associated with the tdh and trh genes,
encoding thermostable direct haemolysin (TDH) and tdh-related haemolysin (TRH),
respectively (15). Open reading frame 8 (ORF8) is considered as a potential factor causing
epidemics and a genetic marker for V. parahaemolyticus O3:K6 strains (23). V.
parahaemolyticus pandemic strains such as O3:K6 strains exhibit a unique toxRS sequence
responsible for the current pandemic in many countries (17).
Increasingly there have been more reports of antibiotic resistance in Vibrio species.
3
Emergence of microbial resistance to multiple drugs is a serious clinical problem in the
treatment, increasing the fatality rate (21).
4
References
1. Alvarado, L.V., and Boehm, K.H., 1989. Virulence factors in motile aeromonads. Spec.
Publ. Eur. Aqua. Soc. 10: 11-12.
2. Angka, S.L., 1990. The pathology of the walking catfish Clarias batrachus (L.)
infected intraperitoneally with Aeromonas hydrophila. Asian Fish. Sci. 3: 343-351.
3. Daniels, N.A., et al., 2000. Vibrio parahaemolyticus infections in the United States,
1973-1998. J. Infect. Dis. 181: 1661-1666.
4. DePaola, A., and Roberts, M.C., 1995. Class D and E tetracycline resistance determinants
in Gram-negative bacteria from catfish ponds. Mol. Cell. Probes 9: 311-313.
5. Esteve, C., Biosca, E.G., and Amaro, C., 1993. Virulence of Aeromonas hydrophila
and some other bacteria isolated from European eels Anguilla anguilla reared in fresh
water. Dis. Aquat. Org. 16: 15-20.
6. Ishimura, K., et al., 1988. Biochemical and biological properties of motile Aeromonas
isolated from aquatic environments. J. Food Hyg. Soc. Japan. 29: 313-319.
7. Jeollabuk-do Province Office, 2008. Available http://www.jeonbuk.go.kr/01kr/
03open_provin/02jb_focus/03news/index2.jsp?MID=C016%3Fbid=do_bodo&mode=
view&cno=13703.
8. Jiangsu Meteorological Bureau, 2004. The requirement of loach is rising year after
year. Nanfang Daily Press Group. Available http://www.jsxnw.gov.cn/ newsfiles/170/
2004-11/1421.shtml [15/11/2006].
9. Joseph, S.W., Colwell, R.R., and Kaper, J.B., 1982. Vibrio parahaemolyticus and
related halophilic Vibrios. Crit. Rev. Microbiol. 10: 77-124.
10. [KFDA] Korea Food and Drug Administration. Annual report of food-born outbreaks,
5
2010. <http://e-stat.kfda.go.kr/> Accessed 12.12.10.
11. Khardori, N., and Fainstein, V., 1988. Aeromonas and Plesiomonas as etiological
agents. Ann. Rev. Microbiol. 42: 395-419.
12. Kim, D.S., Jo, J-Y., and Lee, T-Y., 1994. Induction of triploidy in mud loach
(Misgumus mizofepis) and its effect on gonad development and growth. Aquaculture
120: 263-270.
13. Kim, H.C., Soon, M., and Yu, H.S., 1994. Biological control of vector mosquitos by
the use of fish predators, Moroco oxycephalus and Misgurnus anguillicaudatus in the
laboratory and semi-field rice paddy. Kor. J. Entomol. 24: 269-284.
14. Korea National Statistical office, 2008. The status reports of fishery production in
2008. Available http://index.go.kr/egams/stts/jsp/potal/stts/PO_STTS
_IdxMain.jsp?idx cd=2748&bbs=INDX_001&clas_div=C&rootKey=1.48.0.
15. Lee, J.K., et al., 2008. Occurrence of Vibrio parahaemolyticus in oysters from Korean
retail outlets. Food Control 19: 990-994.
16. Lee, S., et al., 2000. Characterization of Aeromonas hydrophila isolated from rainbow
trouts in Korea. J. Microbiol. 38: 1-7.
17. Matsumoto, C., et al., 2000. Pandemic spread of an O3:K6 clone of Vibrio
parahaemolyticus and emergence of related strains evidenced by arbitrarily primed
PCR and toxRS sequence analyses. J. Clin. Microbiol. 38: 578-585.
18. McGarey, D.J., et al., 1991. The role of motile aeromonads in the fish disease,
ulcerative disease syndrome (UDS). Experientia. 47: 441-444.
19. Miranda, C.D., et al., 2003. Diversity of tetracycline resistance genes in bacteria from
Chilean salmon farms. Antimicrob. Agents Chemother. 47: 883-888.
20. Nawaz, M., et al., 2006. Biochemical and molecular characterization of tetracycline-
6
resistant Aeromonas veronii isolates from catfish. Appl. Environ. Microbiol. 72: 6461-
6466.
21. Okoh, A.I., and Igbinosa, E.O., 2010. Antibiotic susceptibility profiles of some Vibrio
strains isolated from wastewater final effluents in a rural community of the Eastern
Cape Province of South Africa. BMC Microbiol. 10: 143.
22. Ottaviani, D., et al., 2008. First clinical report of pandemic Vibrio parahaemolyticus
O3:K6 infection in Italy. J. Clin. Microbiol. 46: 2144-2145.
23. Parvathi, A., et al., 2006. Molecular characterization of thermostable direct
haemolysin-related haemolysin (TRH)-positive Vibrio parahaemolyticus from oysters
in Mangalore, India. Environ. Microbiol. 8: 997-1004.
24. Rhodes, G., et al., 2000. Distribution of oxytetracycline resistance plasmids between
aeromonads in hospital and aquaculture environments: Implication of Tn1721 in
dissemination of the tetracycline resistance determinant Tet A. Appl. Environ.
Microbiol. 66: 3883-3890.
25. Schmidt, A.S., et al., 2001. Incidence, distribution and spread of tetracycline resistance
determinants and integron encoded antibiotic resistance genes among motile
aeromonads from a fish farming environment. Appl. Environ. Microbiol. 67: 5675-
5682.
26. Schmidt, A.S., et al., 2001. Characterization of class 1 integrons associated with R-
plasmids in clinical Aeromonas salmonicida isolates from various geographical areas.
J. Antimicrob. Chemother. 47: 735-743.
27. Tsukamoto, K., et al., 1993. Phylogenetic relationships of marine bacteria, mainly
members of the family Vibrionaceae, determined on the basis of 16S rRNA sequences.
Inter. J. SysT. Bacteriol. 43: 8-19.
7
Literature Review
A. Aeromonas hydrophila
A.1. Perspective of Aeromonadaceae
The genus Aeromonas (Kingdom, Bacteria; Phylum, Proteobacteria; Class,
Gammaproteobacteria; Order, Aeromonadales; Family, Aeromonadaceae) has undergone a
number of taxonomic and nomenclature revisions in the past. Although originally placed in
the family Vibrionaceae (104), which also included the genera Vibrio, Photobacterium, and
Plesiomonas, subsequent phylogenetic investigations indicated that the genus Aeromonas
is not closely related to vibrios but rather forms a monophyletic unit in the gamma-3
subgroup of the class Proteobacteria (63, 89). These conclusions necessitated the removal
of Aeromonas from the family Vibrionaceae and transfer to a new family, the
Aeromonadaceae (20). Similarly, only five species of Aeromonas were recognized 25 years
ago (47), three of which (A. hydrophila, A. sobria, and A. caviae) existed as phenospecies,
that is, a named species containing multiple DNA groups, the members of which could not
be distinguished from one another by simple biochemical characteristics. Subsequent
systematic investigations have resulted in the number of valid published genomospecies
rising to 14 (50), and it is anticipated that additional species will be described because rare
strains have been identified that do not reside in any established Aeromonas species.
Except for one species, A. salmonicida, they are motile by means of a single polar
flagellum. On the other hand, approximately 30 motile Aeromonas spp. (A.
allosaccharophila, A. aquariorum, A. bestiarum, A. bivalvium, A. cavernicola, A. caviae, A.
8
diversa, A. encheleia, A. enteropelogenes, A. eucrenophila, A. fluvialis, A. hyrophila, A.
jandaei, A. media, A. molluscorum, A. piscicola, A. popoffii, A. rivuli, A. sanarellii, A.
sharmana, A. schubertii, A. simiae, A. taiwanensis, A. tecta, A. trota, A. veronii biovar
sobria, and A. veronii biovar veronii) were identified and those species have been
associated with various human infections such as gastro-enteritis and wound infections,
causing primary and secondary septicemia in immunocompromised persons (31), and have
also been implicated as the causative agents of various fish diseases (49).
A.2. Aeromonadaceae species
From the creation of the genus Aeromonas in 1943 through the mid-1970s, aeromonads
could be broken down roughly into two major groupings, based upon growth
characteristics and other biochemical features (47). The mesophilic group, typified by A.
hydrophila, consisted of motile isolates that grew well at 35 to 37 °C and were associated
with a variety of human infections. The second group, refered to as psychrophilic strains,
caused diseases in fish, wers nonmotile, and had optimal growth temperatures of 22 to
25 °C. This group contained isolates that currently reside within the species A. salmonicida.
Beginning in the mid-1970s and continuing for almost 10 years thereafter, several
groups, including the Institut Pasteur in Paris, the Centers for Disease Control and
Prevention (CDC) in Atlanta, GA, and the Walter Reed Institute of Research in Washington,
DC, spearheaded an effort to redefine the mesophilic group based upon DNA relatedness
studies. Over that span of time, DNA hybridization investigations revealed that multiple
hybridization groups (HGs) existed within each of the recognized mesophilic species (A.
hydrophila, A. sobria, and A. caviae) (30, 84). These unnamed HGs were represented by
reference strains, since in each case they could not be separated unambiguously from each
9
other by simple biochemical means.
A.3. Aeromonas and ecosystems
Aeromonds are essentially ubiquitous in the microbial biosphere. They can be isolated
from virtually every environmental niche where bacterial ecosystems exist. These include
aquatic habitats, fish, foods, domesticated pets, invertebrate species, birds, ticks and insects,
and natural soils, although extensive investigations on the latter subject are lacking. The
vast panorama of environmental sources from which aeromonads can be encountered lends
itself readily to constant exposure and interactions between the genus Aeromonas and
humans.
The relative environmental distributions of Aeromonas species in selected settings, as
currently known, are presented in previous publications. Earlier studies have indicated that
three Aeromonas genomospecies (A. hydrophila, A. caviae, and A. veronii bv. sobria) are
responsible for the vast majority (> 85%) of human infections and clinical isolations
attributed to this genus (46). The same pattern observed clinically appears to repeat itself in
most environmental samples, with A. salmonicida included as a predominant species in
fish and water samples. In some studies, less frequently encountered species have been
found to predominate in environmental samples, such as A. schubertii in organic
vegetables (67). For newly described species such as A. aquariorum and A. tecta, no data
exist on their relative distributions in the environment outside their initial taxonomic
description, and extremely limited data are available on many other taxa described since
2004. Finally, the techniques and methods used to identify Aeromonas isolates to the
species level vary considerably from one study to the next.
10
A.4. Virulence factors of Aeromonas
Virulence of Aeromonas sp. is multifactorial and not completely understood (29).
Aeromonas sp. have been reported to elaborate exotoxins (hemolysins, cytotoxins, and
enterotoxins), hemagglutinins, adhesins, several hydrolytic enzymes, and invade tissue in
culture (10, 36, 45, 57). The hemolysin produced by some Aeromonas sp. (also known as
aerolysin) has been shown to have both hemolytic and enterotoxic activity (5, 16). Burke et
al. (9) found that 97% of the hemolysin-producing strains were able to secrete enterotoxins.
Other investigators also reported a correlation between hemolysin and cytotoxin
production (82). The hemolytic enterotoxin shares significant homology with the cytotoxic
enterotoxin (Act), and two cytotonic toxins (Alt and Ast) (19). Rahim et al., (85) tested 32
act gene probe-positive and 31 randomly selected act gene probe-negative Aeromonas
isolates for enterotoxicity in a suckling mice assay (SMA), for haemolytic activity on
sheep blood agar plates, for the presence of CAMP-like factors, and for cytotoxicity in a
Vero cell line. This study indicated the role of Act in the pathogenesis of Aeromonas
infections and that the enterotoxic potential of Aeromonas sp. could be assessed by simply
performing a CAMP-haemolysin assay.
Enterotoxigenic isolates of A. hydrophila showed hemagglutination (HA) which was
not sensitive to mannose (i.e. mannose-resistant [MRHA]) and fucose, but Aeromonas
strains that were HA-sensitive to mannose or showed no hemagglutination (NHA) were
non-toxic strains of A. caviae commonly isolated from nondiarrhoeal infection ot the
environment (10). In enteric bacteria, hemagglutination of erythrocytes is associated with
the ability to adhere to human epitherial cells.
11
A.5. Aeromonas and aquatic environments
Groundbreaking studies conducted over 30 years ago by Terry Hazen and associates
identified viable Aeromonas in 135 of 147 (91.8%) natural aquatic habitats sampled in the
United States and Puerto Rico (38). Aeromonas numbers were higher in lotic than in lentic
systems and were higher in thermal gradients ranging from 25 to 35 °C (38, 39). A.
hydrophila grew over a wide range of temperatures, conductivities, pHs, and turbidities,
with only those habitats with extreme ranges of these parameters (extremely saline
environments, thermal springs, and highly polluted waters) failing to yield aeromonads.
Today, the genus Aeromonas is considered to be almost synonymous with water and
aquatic environments, being isolated from rivers, lakes, ponds, seawaters (estuaries),
drinking water, groundwater, wastewater, and sewage in various stages of treatment.
Concentrations of aeromonads in these sites have been reported to vary from lows of <1
CFU/ml (groundwater, drinking water, and seawater) to highs of 108 CFU/ml or more, in
crude sewage or domestic sewage sludge (41). Although primarily a freshwater resident,
Aeromonas species can be recovered from the epipelagic layer (<200 m) of the ocean (as
opposed to benthic regions), most often in estuaries, existing as free-living bacteria or in
association with crustaceans. Estuaries are ideally suited for aeromonads, since salinity
concentrations are substantially lower there than in the deeper (benthic) regions of the
ocean. One study from the Italian coast found aeromonad numbers varying from 102 to 106
CFU per 100 ml throughout the year (32).
12
A.6. Aeromonas and fish diseases
The role of aeromonads as a causative agent of fish diseases has been known for
decades, longer than their comparable role in causing systemic illnesses in humans. Two
major groups of fish diseases are recognized. A. salmonicida causes fish furunculosis,
particularly in salmonids. The disease has several presentations, ranging from an acute
form characterized by septicemia with accompanying hemorrhages at the bases of fins,
inappetence, and melanosis to a subacute to chronic variety in order fish, consisting of
lethargy, slight exophthalmia, and hemorrhaging in muscle and internal organ (6).
Mesophilic species (A. hydrophila and A. veronii) cause a similar assortment of diseases in
fish, including motile Aeromonas septicemia (hemorrhagic septicemia) in carp, tilapia,
perch, catfish, and salmon, red sore disease in bass and carp, and ulcerative infections in
catfish, cod, carp, and goby (49). Mesophilic Aeromonas species, most notably A.
hydrophila, have been linked to major die-offs and fish kills around the globe over the past
decade, resulting in enormous economic losses. These die-offs included over 25,000
common carp in the St. Lawrence River in 2001 (71), 820 tons of goldfish in Indonesia in
2002, resulting in a 37.5 million dollars loss, and a catfish die-off in Minnesota and North
Dakota in 2007. In many of these instances, Aeromonas species were sole or copathogens
causing invasive secondary infections in immunosuppressed fish due to spawning or
environmental triggers, such as high temperatures or low water levels.
A.7. Antimicrobial resistance
In the last two decade, high rates of resistance to commonly used, cheap oral antibiotics
among enteric pathogens has been reported from several developing countries (86, 91,
103). The same story can be said for Aeromonas sp., particularly those isolated from
13
clinical sources and to a lesser extent from foods and water. The ease of which
antimicrobial agents can be obtained in these countries has been blamed for this problem
(2, 90). High resistance rates to antimicrobial agents appear to be common among
aeromonads isolated from fish in developing countries. Antimicrobial agents are used
extensively in fish farms to treat and prevent fish diseases and also as feed additives. Such
practice has been shown to increase drug resistant bacteria as well as R plasmids (37, 108).
However, variation in the resistance rates of aeromonads to different antimicrobial agents
in different developing countries can be observed. Such differences in the frequency of
resistance may well be related to the source of the Aeromonas isolates and the frequency
and type of antimicrobial agents prescribed for treating Aeromonas infections in different
geographical areas (97).
Resistance of most aeromonads to ampicillin is generally considered to be intrinsic or
chromosomal mediated (4). Several studies have shown that patients taking ampicillin for
reasons other than diarrhoea may predispose them to infection with Aeromonas (33, 72).
Moyer (72) reported that for the susceptible host, antibiotic therapy, and drinking of
untreated water are two significant risk factors for infection with Aeromonas. However,
gastrointestinal infections with Aeromonas are generally self-limiting. Although treatment
of patients with symptoms of infectious diarrhea with antibiotics remains controversial,
antimicrobial therapy should be initiated for those who are severely ill and for patients
with risk factors for extraintestinal spread of infection after obtaining appropriate blood
and fecal cultures (40). The current accepted treatment of all acute infectious diarrhoeal
diseases is rehydroation, antibiotic treatment, and nutritional therapy (90).
14
B. Vibrio parahaemolyticus
B.1. Perspective of Vibrio
The genus Vibrio (Kingdom, Bacteria; Phylum, Proteobacteria; Class,
Gammaproteobacteria; Order, Vibrionales; Family, Vibrionaceae) has played a significant
role in human history. Outbreaks of cholera, caused by Vibrio cholerae, can be traced back
in time to early recorded descriptions of enteric infections. Indeed, the path of human
history has been influenced significantly by this organism (83, 107, 109). First described
by Pacini (80) while he was a medical student in Italy and at a time when the germ theory
of disease was in dispute, V. cholerae was subsequently identified and described in greater
detail by Robert Koch (54, 55), to whom credit for the discovery of the causative agent of
cholera traditionally has been given.
The germ theory of disease was developed in the 19th century, based on the British
queen’s physician John Snow’s tracing an 1849 cholera outbreak to a single contaminated
well in the Broad Street area of central London; it remains a canonical example of
epidemiology. Snow’s demonstration was an important mile-stone in public health,
correctly identifying the fecal-oral route to human infection and offering powerful
arguments for the germ theory (96). Many advances in the prevention and treatment of
infectious diseases during the latter half of the 19th century and the first half of the 20th
century follow directly from the acceptance of Snow’s point of view.
The vibrios have also received the attention of marine microbiologists who observed
that the readily cultured bacterial populations in near-shore waters and those associated
with fish and shellfish were predominantly Vibrio spp. For example, the “gut group”
vibrios were described by Liston (58, 59), working at the Marine Laboratory in Aberdeen,
15
Scotland. Fish diseases caused by vibrios have been reviewed extensively by marine
investigators and, among the many fish pathogens, Vibrio anguillarum has been recognized
historically as a major pathogen of marine animals.
B.2. Viewpoint of Vibrio parahaemolyticus
Vibrio parahaemolyticus is a Gram-negative, halophilic asporogenous rod that is
straight or has a sinple, rigid curve. It has a single polar flagellum and is motile when
grown in liquid medium (7). This bacterium is a human pathogen that occurs naturally in
the marine environments and frequently isolated from a variety of seafoods including
codfish, sardine, mackerel, flounder, clam, octopus, shrimp, crab, lobster, crawfish, scallop,
and oyster (60). Consumption of raw or undercooked seafood, particularly shellfish,
contaminated with V. parahaemolyticus may lead to development of acute gastroenteritis
characterized by diarrhea, headache, vomiting, nausea, abdominal cramps, and low fever.
This bacterium is recognized as the leading cause of human gastroenteritis associated with
seafood consumption in the United States and an important seafood-borne pathogen
throughout the world (53).
Although the gastroenteritis caused by V. parahaemolyticus infection is often self-
limited, the infection may cause septicemia that is life-threatening to people having
underlying medical conditions such as liver disease or immune disorders. Two deaths were
among three cases of wound infections caused by V. parahaemolyticus in Louisiana and
Mississippi after Hurricane Katrina in 2005 (14).
16
B.3. Occurrence of Vibrio parahaemolyticus
The distribution of V. parahaemolyticus in the marine environments is known to relate
to the water temperatures. Studies have shown that the organism was rarely detected in
seawater until water temperatures rose to 15 °C or higher. Ecological study of V.
parahaemolyticus in the Chesapeake Bay of Maryland found that V. parahaemolyticus
survived in sediment during the winter and was released from sediment into water column
when water temperatures rose to 14 °C in late spring or early summer (51). Another survey
of nine U.S. coastal states conducted between 1984 and 1985 reported an average low
density of V. parahaemolyticus in seawater when water temperatures dropped below 16 °C
(26). However, the densities of V. parahaemolyticus in seawater could increase when water
temperatures increased to around 25 °C (26, 51). A recent study of occurrence of V.
parahaemolyticus in Oregon oyster-growing environments between November 2002 and
October 2003 also found a positive correlation between V. parahaemolyticus in seawater
and water temperatures with the highest populations of V. parahaemolyticus in water being
detected in the summer months (24).
The degree of V. parahaemolyticus contamination in raw shellfish is also known to
relate to the water temperatures. Therefore, it is more likely to detect V. parahaemolyticus
in oysters harvested in the spring and the summer than in the winter. V. parahaemolyticus
can multiply rapidly in oysters upon exposure of elevated temperatures. Studies have
shown that populations of V. parahaemolyticus in unrefrigerated oysters could increase
rapidly to 50-790 folds of its original level within 24 h of harvest if oysters were exposed
to 26 °C (35). A survey of 370 lots of oysters sampled from restaurants, oyster bars, retail
and wholesale seafood markets throughout the US between June 1998 and July 1999 found
a seasonal distribution of V. parahaemolyticus in market oysters with high densities being
17
detected in the summer months (21).
B.4. Incidence of V. parahaemolyticus food poisoning
V. parahaemolyticus was first recognized as a cause of food-borne illness on Osaka,
Japan in 1951 (23). It caused a major outbreak of 272 illness and 20 deaths associated with
consumption of sardines. Since then, V. parahaemolyticus has been reported to account for
20-30% of food poisoning cases in Japan (1) and identified as a common cause of seafood-
borne illness in many Asian countries (17, 25, 110). V. parahaemolyticus was the leading
cause of food poisoning (1710 incidents, 24,373 cases) in Japan between 1996 and 1998
(44) and accounted for 69% (1028 cases) of total bacterial foodborne outbreaks (1495
cases) reported in Taiwan between 1981 and 2003 (3) and 31.1% of 5770 foodborne
outbreaks occurred in China between 1991 and 2001 (61).
In contrast to Asian countries, V. parahaemolyticus infections are rarely reported in
European countries. However, sporadic outbreaks have been reported in countries such as
Spain and France. Eight cases of V. parahaemolyticus gastroenteritis related to fish and
shellfish consumption were reported in Spain in 1989 (70). An outbreak of 64 illnesses
associated with raw oysters consumption occurred in 1999 in Galicia, Spain (62). A serious
outbreak affecting 44 patients associated with consumption of shrimps imported from Asia
accurred in France in 1997 (88). A more recent outbreak involving 80 illnesses of V.
parahaemolyticus infection among guests attending weddings in one restaurant was
reported in Spain in July 2004 (64). Epidemiological investigation associated the outbreak
with consumption of boiled crab that had been processed under unhealthy conditions.
V. parahaemolyticus was first identified as an etiological agent in the US in 1971 after
three outbreaks of 425 cases of gastroenteritis associated with consumption of improperly
18
cooked crabs occurred in Maryland (69). Since then, sporadic outbreaks of V.
parahaemolyticus infections related to consumption of raw shellfish or cooked seafood
were reported throughout the US coastal regions. Between 1973 and 1998, approximately
40 outbreaks of V. parahaemolyticus infections were reported to the Centers for Disease
Control and Prevention (CDC) (22). Among them, four major outbreaks involving more
than 700 cases of illness associated with raw oyster consumption occurred in the Gulf
Coast, Pacific Northwest, and Atlantic Northeast regions between 1997 and 1998. In the
summer of 1997, 209 cases (including one death) of V. parahaemolyticus infections
associated with raw oyster consumption occurred in the Pacific Northwest (Oregon,
Washington, California and British Columbia of Canada) (12). In 1998, two outbreaks
occurred in Washington (43 cases) and Texas (416 cases) were associated with
consumption of raw oyster (27). In addition, a small outbreak of eight cases of V.
parahaemolyticus infections was reported in Connecticut, New Jersey, and New York
between July and September in 1998 as a result of eating oysters and clams harvested at
Long Island Sound of New York (13). Recently, 14 passengers on board a cruise ship in
Alaska developed gastroenteritis after eating raw oysters produced in Alaska in the
summer of 2004 (66). More recently, an outbreak of V. parahaemolyticus involving 177
cases occurred in the summer of 2006 was linked to contaminated oysters harvested in
Washington and British Columbia (15). The occurrence of these outbreaks indicates that
contamination of V. parahaemolyticus in oysters is a safety concern in the US.
19
B.5. Virulence factors
It is known that most strains of V. parahaemolyticus isolated from the environment or
seafood are not pathogenic (77). Clinical strains of V. parahaemolyticus are differentiated
from environmental strains by their ability to produce a thermostable direct hemolysin
(TDH), an enzyme that can lyse red blood cells on Wagatsuma blood agar plates. The
hemolytic activity of TDH, named the Kanagawa phenomenon, has been reported to be
commonly associated with strains isolated from humans with gastroenteritis but were
rarely observed in environmental isolates (48). Therefore, the TDH has been recognized
the major virulence factor of V. parahaemolyticus (68, 100).
Despite epidemiological investigations revealed a strong tie between the Kanagawa
phenomenon (KP) and the pathogenicity of V. parahaemolyticus, KP-negative strains that
did not produce TDH but a TDH-related hemolysin (TRH) had been isolated from outbreak
patients (42, 43). Shirai et al. (92) examined 215 clinical strains of V. parahaemolyticus
isolated from patients with diarrhea for presence of genes encoding TDH (tdh) and TRH
(trh) and found that 52 strains (24.3%) carried only the trh gene. These results indicate that
TRH is also a virulence factor of V. parahaemolyticus. The genes encoding TDH (tdh) and
TRH (trh) have been cloned and sequenced (52, 75, 101). Oligonucleotide probes for both
tdh and trh genes have been developed for detection of virulent strains of V.
parahaemolyticus (52, 76).
B.6. Pandemic strains of V. parahaemolyticus
Most outbreaks of V. parahaemolyticus infections were caused by V. parahaemolyticus
of diverse serotypes. However, increased incidences of gastroenteritis caused by V.
parahaemolyticus serotype O3:K6 have been reported in many countries since 1996 (18,
20
34, 64, 106). This serovar was first identified during a hospital-based active surveillance
study of V. parahaemolyticus infections in Calcutta, India between 1994 and 1996 (79).
The survet identified a sudden increase in incidences associated with this serovar, which
accountered for 63% of total V. parahaemolyticus strains isolated from patients in Calcutta
between September 1996 and April 1997. This highly virulence strain was subsequently
recovered at a high rate in other Southeast Asian countries and was isolated from travelers
arriving in Japan from various countries in the Southeast Asia (18, 79 106).
V. parahaemolyticus O3:K6 was first identified in the US in 1998 and caused the largest
outbreak (416 person) associated with oyster consumption in the US history (23). The
same serovar wa later involved in another outbreak related to shellfish consumption in
Connecticut, New Jersey, and New York (13). Since then, a pandemic spread of this clone
to other continents has been reported. In 2004, V. parahaemolyticus O3:K6 was isolated
from victims of outbreaks occurred in Chile (34) and Spain (64). The isolation of the
O3:K6 strain from US outbreaks raised concern about increased risks of V.
parahaemolyticus infections from US consumption. However, this serovar has not been
linked to illness resulted from consuming raw oysters in the US since 1999.
21
C. Bacteriophage (phage)
C.1. General description
Phages are viruses that invade bacterial cells and, in the case of lytic phages, disrupt
bacterial metabolism and cause the bacterium to lyse. They were discovered by Ernest
Hankin (1896) and Frederick Twort (1915) who described their antibacterial activity.
However, Felix d’Herelle (1919) was probably the first scientist who used phages as a
therapy to treat severe dysentery. At that time, several companies then actively started up
the commercial production of phages against various bacterial pathogens for human use.
However, phage production was quickly displaced by the discovery of antibiotics in most
of the Western world. Nevertheless, phage therapy is still an on-going practice in Eastern
Europe and countries from the former Soviet Union. Several institutions in these countries
have been involved in phage therapy research and production, with activities centralized at
the Eliava Institute of Bacteriophage, Microbiology and Virology (Tbilisi, Georgia) and the
Hirszfeld Institute of Immunology and Experimental Therapy (Wroclaw, Poland). Their
work in this field has recently been extensively reviewed (56).
The current threat of antibiotic-resistant bacteria has renewed the interest in exploring
phages as biocontrol agents in Western countries (65, 98). In fact, some products based on
phages are already commercially available (‘PhageBioderm’, ‘Bacteriophagum Intestinalis
Liquidum’, ‘Pyobacteriophagum Liquidum’). Additionally, some care centers are
particularly specialized in phage therapy (for example, Southwest Regional Wound Care
Center, Texas).
Besides phage therapy, the use of phages as antimicrobial agents and tools for detecting
pathogens in feed and foodstuffs is also expanding with several campanies having been
22
created recently (Table I).
Table I. Companies involved in phage application.
Company Country Website
BIOphage PHARMA inc. Canada http://www.biophagepharma.net
BIOPHARMA LIMITED Georgia http://www.biopharmservices.com
CJ CheilJedang Korea http://www.cj.co.kr
CTCBIO INC. Korea http://www.ctcbio.com
MICREOS Food Safety Netherlands http://www.ebifoodsafety.com
Exponential Biotherapies Inc. European Union http://www.expobio.com
GangaGen, Inc. European Union http://www.gangagen.com
Hexal-gentech Germany http://www.hexal-gentech.com
Intralytix, Inc. European Union http://www.intralytix.com
Komipharm International Co. Korea http://komilab.com
Novolytics United Kingdom http://www.novolytics.co.uk
Omnilytics, Inc. European Union http://www.phage.com
Phage Biotech Ltd. Israel http://www.phage-biotech.com
Fields of application comprise of water and food safety, agriculture and animal health.
An example is OmniLytics, Inc. that gained US Environmental Protection Agency approval
for the use of its product AgriPhage against plant pathogenic bacteria. In food
manufacturing industry, EBI Food Safety recently marketed ListexTM P100 for controlling
Listeria in meat and cheese products (11). In August 2006, the US Food and Drug
Administration (FDA) approved the use of a phage preparation targeting Listeria, LMP
102 (Intralytix, Inc.), in ready-to-eat meat and poulty products.
23
C.2. Phage application in aquaculture
Cultured fish and shellfish, like other animals and humans, are constantly threatened by
microbial attacks. Although chemotherapy is a rapid and effective method to treat or
prevent bacterial infections, frequent use of chemotherapeutic agents has allowed drug-
resistant strains of bacteria to develop. In particular, this problem in chemotherapy may be
serious in fishery industries (74). Needless to say, vaccination is an ideal method for
preventing infectious diseases, but commercially available vaccines are still very limited in
the aquaculture field. This is partly due to the fact that many different kinds of infectious
diseases occur locally in a variety of fish and shellfish species. Studies on biological
control such as probiotics have been sporadically reported in the field of fish pathology (28,
78, 105); however, they involve substantial difficulties in scientific demonstration of the
causal sequence, as mentioned in human use of probiotics (102). In view of a scientific
demonstration of phage treatment, the causal effect of phages in successful phage therapy
can be definitively proven by confirming an increase in phage particles in the number or
the presence of phages in the survivors, which is the result of the death of host bacterial
cells. The feasibility of this demonstration distinguishes phage treatment from other
biological controls, which fail to utilize scientific methodology in demonstrating causal
relationships. Under these circumstances, phages, as specific pathogen killers, could be
attractive agents for controlling fish bacterial infections. Phages of some fish pathogenic
bacteria, such as Aeromonas salmonicida, A. hydrophila, Edwardsiella tarda, Lactococcus
garvieae, Pseudomonas plecoglossicida, and Yersinia ruckeri, have been reported (73, 74,
81).
24
C.3. Phage therapy
From the continued observations and experiences treating patients, d’Herelle quickly
realized that not all phage preparations were effective and that care had to be taken both
when preparing and when applying phages. At the same time, the advent of effective
chemical antibiotics in the 1930s and 1940s led to the therapeutic use of phages in the West
being curtailed. However, clinical use continued in the countries of the former Soviet bloc.
Interest in phage therapy was reignited by increasing concern over antibiotic resistance
and also by publication of successful results achieved by of Smith et al. (93, 94, 95), even
demonstrating the apparently superior efficacy of phage therapy compared to antibiotics in
a mouse model of E. coli infection (93). The advent of multi-drug resistant pathogens has
forced the re-examination of phage therapy, with work being carried out to modern
regulatory standards.
A large amount of indicative data supporting the effectiveness of phage therapy is
available from studies involving human patients in Eastern Europe, with few reported
adverse events. This evidence for safety, while not up to current regulatory standards, is
further supported by the exposure of humans to high levels of phages via everyday
activities because of the ubiquitous nature of phages in the environment.
Human safety trials have also been performed with increasing frequency including
extensive safety trials undertaken on Staphage Lysate by Delmont Laboratories (USA) (98).
This product, which contains high concentrations of antistaphylococcal phages, was
administered to humans intranasally, topically, orally, subcutaneously, and intravenously.
In over 12 years of use in humans, only minor side effects were observed (98, 99). In a
formal safety study, Harold Brussow based at the Nestle Research Center, Switzerland,
demonstrated no safety concerns when phages targeting E. coli were administered to
25
human volunteers (8).
An FDA-approved phase I physician-led trial has been completed at a wound care
center in Lubbock, Texas (87). Jusing a mixture of phages targeting Pseudomonas
aeruginosa, Staphylococcus aureus, and E. coli and also showed no safety concerns.
D. References
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parahaemolyticus in the Seto-Inland Sea, Japan. FEMS Microbiol. Lett. 208: 83-87.
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5. Asao, T., et al., 1984. Purification and some properties of Aeromonas hydrophila
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parahaemolyticus infections associated with eating raw oysters and clams harvested
from Long Island Sound - Connecticut, New Jersey and New York, 1998. Morb.
Mortal. Wkly. Rep. 48: 48-51.
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Hurricane Katrina - Multiple States, August-September 2005. Morb. Mortal. Wkly.
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infections associated with consumption of raw shellfish - three States, 2006. Morb.
Mortal. Wkly. Rep. 55: 1-2.
16. Chakraborty, T., et al., 1986. Cloning, expression, and mapping of the Aeromonas
hydrophila aerolysin gene determinant in Escherichia coli K-12. J. Bacteriol. 167:
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17. Chen, S., Liu, S., and Zhang, L., 1991. Occurrence of Vibrio para-haemolyticus in
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unusually high incidence of food-borne disease outbreaks in Taiwan from 1996 to
1999. J. Clin. Microbiol. 38: 4621-4625.
19. Chopra, A.K., and Houston, C.W., 1999. Enterotoxins in Aeromonas asso-ciated
gastroenteritis. Microbes Infect. 1: 1129-1137.
20. Colwell, R.R., MacDonell, M.T., De Ley, J., 1986. Proposal to recognize the family
Aeromonadaceae. Int. J. Syst. Bacteriol. 36: 473-477.
21. Cook, D.W., et al., 2002. Vibrio vulnificus and Vibrio parahaemolyticus in US retail
shell oysters: a national survey from June 1998 to July 1999. J. Food Prot. 65: 79-87.
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37
Chapter I
Protective effects of the Aeromonas phages pAh1-C and
pAh6-C against mass mortality of the cyprinid loach
(Misgurnus anguillicaudatus) caused by Aeromonas
hydrophila
Abstract
To investigate methods to control the mass mortality of cyprinid loaches (Misgurnus
anguillicaudatus) caused by multiple-antibiotic-resistant Aeromonas hydrophila on a
private fish farm in Korea, bacteriophages (phages), designated pAh1-C and pAh6-C, were
isolated from the Han River in Seoul. The two isolated phages were morphologically
classified as Myoviridae and showed similar infection patterns for A. hydrophila isolates.
The two phages possessed approximately 55 kb (pAh1-C) and 58 kb (pAh6-C) of double-
stranded genomic DNA, and their gDNAs showed different restriction endonuclease
digestion patterns. Both phages showed efficient bacteriolytic activity against fish-
pathogenic A. hydrophila from loaches. The latent periods of the phages were estimated to
be approximately 30 min (pAh1-C) and 20 min (pAh6-C), while the burst sizes were 60
PFU/cell (pAh1-C) and 10 PFU/cell (pAh6-C). The phages proved to be efficient in the
inhibition of bacterial growth, as demonstrated by their in vitro bactericidal effects.
Additionally, a single administration of either phage to cyprinid loaches resulted in
noticeable protective effects, with increased survival rates against A. hydrophila infection.
These results suggest that the phages pAh1-C and pAh6-C constitute potential therapeutic
38
agents for the treatment of A. hydrophila infection in fish.
Keywords: Cyprinid loach, Misgurnus anguillicaudatus; Multiple-antibiotic-resistant;
Aeromonas hydrophila; Bacteriophage; Therapeutic agent.
39
1.1. Introduction
The loach (Misgurnus spp.) is a member of the Cobitidae family (Lacepede, 1803) and
inhabits freshwater systems throughout the world (17). Two species of loaches, the mud
loach (M. mizolepis) and the cyprinid loach (Misgurnus anguillicaudatus), are cultured for
a variety of uses, primarily for food, in Korea (16). The annual demand for loaches in
Korea is one of the highest for fresh-water fish, due to their high nutritional value and use
in both folk medicine and Buddhist ceremonies.
Aeromonas spp. are primary organisms of normal aquatic microflora, and recently, there
has been an increasing appreciation of their role as waterborne pathogens of fish and
humans (31). Aeromonas hydrophila is a motile aeromonad that can cause disease in fish,
resulting in high rates of mortality (2, 3, 8, 12, 15, 22). There has been an increasing
incidence of antimicrobial resistance among Aeromonas sp. isolated from aquaculture
environments (15, 33, 36, 37). Multiple-antibiotic-resistant A. hydrophila exists in
aquaculture systems and contributes to the high rate of mortality within the fish industry in
Korea (15). Although the majority of the loach population in Korea is cultured and A.
hydrophila is one of the main causes of mass mortality in these fish, no effective method
has been proposed for the control of A. hydrophila infection in aquaculture, except for the
application of additional antibiotics.
The use of phages has been proposed for the treatment of infectious disease, and several
studies have shown that phages can be used successfully for the treatment of bacterial
infections in both humans and animals (4, 6, 20, 21, 40). Phage therapy is advantageous in
that it is natural and relatively inexpensive, and so far, no serious or irreversible side
effects have been described (10, 38). Although phage studies of some fish-pathogenic
40
bacteria have been conducted (23, 24, 25, 26, 30, 34, 41), there have been few attempts to
use phages to control bacterial infections in fish (29). This study aimed to isolate and
characterize A. hydrophila-specific phages and to evaluate their therapeutic potential
during experimental infections of loaches with A. hydrophila.
1.2. Materials and methods
1.2.1. Bacterial strains and culture media
The bacterial strains (n = 17) used in this study included 5 A. hydrophila strains (3 fish-
pathogenic clinical isolates and 2 environmental isolates), 4 strains of other Aeromonas spp.
(3 fish-pathogenic clinical isolates and 1 environmental isolate), and 8 additional strains of
different genera; these strains are listed in Table 1.1. Laboratory stock strains (Table 1.1)
were used to determine the host range of the isolated phages. Tryptic soy broth (TSB) and
tryptic soy agar (TSA) were used for bacterial culture and phage PFU assays.
1.2.2. Phage isolation and host range
Phages were isolated from natural water of the Han River in May 2010 by the
enrichment technique. A water sample (100 ml) was filtered through a 0.45 μm pore-size
membrane filter, and the filtrate was mixed with 100 ml of TSB containing fish-pathogenic
A. hydrophila (JUNAH) as the indicator organism. After 24 h of growth at 25 °C with
gentle agitation, the culture was centrifuged (13,000 rpm/10 min) and filtered again. The
phage activity in the supernatant was then detected with a spot assay (5). The phages were
further purified by a CsCl continuous density gradient (35).
The plaques were classified into the following three categories according to the degree
41
of clarity: clear, turbid, and no reaction. A plaque-forming unit (PFU) assay was performed
using the double-agar-layer method (30). The number of PFU was determined after 24 h of
incubation at 25 °C, and the efficiency of plating (EOP) was also calculated. The host
ranges of the two phages were determined by the double-agar-layer method.
1.2.3. Electron microscopy
Phage particles were negatively stained with 2% uranyl acetate, and electron
micrographs were captured with a JEM 1010 transmission electron microscope (JEOL,
Akishima, Japan) and a LIBRA 120 energy-filtering transmission electron microscope
(Carl Zeiss, Germany). The phage size was considered to be the average of 5-7
independent measurements.
1.2.4. One-step growth
One-step growth curves were obtained for pAh1-C and pAh6-C according to the
method of Verma et al. (39). The optical density (OD) of mid-exponential host bacterial
cultures (A. hydrophila JUNAH) at 600 nm was adjusted to a corresponding cell density of
8.0×106 CFU/ml. The phage suspension (10 μl) was added to 10 ml of the bacterial culture
to obtain a multiplicity of infection (MOI) of 0.001. The phages were allowed to adsorb for
5 min at room temperature. The mixture was then centrifuged (13,000 rpm/3min), and the
resulting pellet was resuspended in 20 ml of TSB. Samples (100 μl each) were collected at
5 min intervals and subjected to phage titration.
42
1.2.5. Phage DNA isolation and restriction endonuclease analysis
Phage DNA was extracted from pure stocks as previously described (35) and subjected
to nuclease treatment using DNase I, RNase A, and Mung bean nuclease according to the
supplier’s instructions (Takara Bio Inc., Japan). In addition, the size estimation and
restriction analysis of phage DNA were performed by pulsed-field gel electrophoresis, as
described previously by Kim et al. (19).
1.2.6. Phage structural protein analysis
Phage structural proteins were analyzed by SDS-PAGE as previously described (19).
After electrophoresis, the protein bands were visualized by staining the gel with Coomassie
blue R-250 and identified by liquid chromatography-tandem mass spectrometry (LC-
MS/MS) at the National Instrumentation Center for Environmental Management (NICEM)
at Seoul National University.
1.2.7. Phage stability test
Phage stability tests were conducted as described elsewhere (39), with some
modifications. Briefly, to evaluate the stability of the phages in organic solvents, equal
volumes of phage solution (105 PFU/ml) and appropriate organic solvents, such as
chloroform, ethanol, and ether, were mixed and incubated at 25 °C for 1 h. To evaluate the
stability of the phages at different pH values, the pH of the TSB was adjusted with either 1
M HCl or 1 M NaOH to obtain solutions with pH values of 3, 5, 7, 9, and 11. The pAh1-C
and pAh6-C phage suspensions were adjusted to obtain a final concentration of 107
PFU/ml and were subsequently incubated at 25 °C for 1 h. Phage suspensions maintained
at pH 7 were used as controls. The stability of the phages at various temperatures (20, 25,
43
30, 37, 50, and 65 °C) was determined by incubating the phages (105 PFU/ml) at the
respective temperatures for 1 h. After incubation, the phage titer was estimated by the
double-agar-layer method.
1.2.8. Host cell lysis test
The bactericidal effect of the phages on A. hydrophila (JUNAH) was determined by
measuring the viable bacterial counts throughout the incubation period. TSB (20 ml) was
inoculated with 0.1 ml of an overnight A. hydrophila (JUNAH) culture and incubated at
25 °C with shaking until the early exponential phase was reached (OD600 nm = 0.1) (8.0×106
CFU/ml). The phages were added at the indicated MOIs (multiplicity of infection), and the
changes in OD were monitored for 24 h.
1.2.9. Preparation of phage-resistant A. hydrophila variants
An A. hydrophila (JUNAH) culture was treated with an undiluted suspension (1010
PFU/ml) of the pAh1-C and pAh6-C phages. Colonies appearing on the plate after 2 days
of incubation at 25 °C were purified on TSA, and the selection for phage-resistant variants
was repeated. Finally, the cultures that produced no PFU after the addition of 1010 PFU/ml
of each phage were used as phage-resistant variants.
1.2.10. Phage treatment of infected fish
A total of 2,400 healthy cyprinid loaches (Misgurnus anguillicaudatus) (average
weight: 14.98 g) were divided into 60 groups in 7 L fiber plastic tanks at 25 ± 1 °C. In the
1st experiment, all fish were infected intraperitoneally (IP) with A. hydrophila (JUNAH),
and the treatment groups were immediately injected with pAh1-C and pAh6-C. The
44
concentrations of bacteria injected were 2.6×106 CFU/fish for the 1st trial and 2.6×107
CFU/fish for the 2nd trial. The concentrations of phages used were 3.0×107 PFU/fish for
pAh1-C and 1.7×107 PFU/fish for pAh6-C. In the 2nd experiment, all conditions were the
same as in the 1st experiment, but the treatment groups were fed pellets (0.5% based on
body weight) that had been impregnated with the phage suspensions containing pAh1-C
and pAh6-C.
Six groups of 40 loaches were used to study the virulence of the phage-resistant A.
hydrophila (JUNAH) variants. The mortality rates of the fish were recorded daily for 7
days, and the kidneys of both the dead and surviving fish were subjected to a bacterial
isolation study, as previously described (15). Every experiment was performed on three
separate occasions, and the results shown represent the mean of these three observations ±
the standard deviation (SD). All animal experiments were performed according to the
guidelines of the Animal Ethical Committee of Seoul National University, Seoul, Republic
of Korea.
1.2.11. Statistical analysis
The experimental results were analyzed for statistically significant differences using
Student’s t-test. A P value of less than 0.05 was accepted as statistically significant. The
SPSS statistical software package version 13.0 (SPSS, Inc., Chicago, IL) was used for all
statistical analyses.
45
1.3. Results
1.3.1. Morphology, host range, and one-step growth patterns of the Aeromonas phages
pAh1-C and pAh6-C
From May 2009 to December 2010, 22 phages were isolated from water samples from
the Han River in Seoul. From the isolated phages, two Aeromonas phages, designated
pAh1-C and pAh6-C, produced clear plaques and were selected for further studies (Figure
1.1). pAh1-C formed small plaques (average diameter: 1.6 mm) in A. hydrophila JUNAH,
while pAh6-C formed large plaques (average diameter: 3.8 mm). Both phages had
isometric heads, necks, and contractile tails, with tail fibers. Based on their morphologies
and according to the classification system of Ackermann (1), both phages belong to the
family Myoviridae (Figure 1.1). The tail lengths were 113 ± 6 nm for pAh1-C and 124 ± 6
nm for pAh6-C (mean ± SD) (n = 10), while the widths were 14 ± 3 nm for pAh1-C and 18
± 1 nm for pAh6-C (n = 10). The head diameters were 41 ± 3 nm for pAh1-C and 63 ± 3
nm for pAh6-C (n = 10).
To evaluate the host ranges of pAh1-C and pAh6-C, they were tested on various
Aeromonas spp. and A. hydrophila strains. Among the 9 Aeromonas sp. tested, the two
phages inhibited the growth of all A. hydrophila strains (n = 5), with pAh1-C producing
clear plaques on two strains and pAh6-C producing clear plaques on three strains. The EOP
values varied among the Aeromonas spp., and no strain showed a value higher than that of
the indicator host strain JUNAH (Table 1.1). pAh1-C and pAh6-C failed to lyse any of the
other 8 bacterial strains used in this study. Additionally, a one-step growth test showed that
the latent periods were approximately 30 min (pAh1-C) and 20 min (pAh6-C) and that the
burst sizes were 60 PFU/cell (pAh1-C) and 10 PFU/cell (pAh6-C) (Figure 1.2).
46
1.3.2. Genomic and proteomic characteristics of the Aeromonas phages pAh1-C and pAh6-
C
The DNA of both phages was completely digested by DNase I but not by RNase A or
Mung bean nuclease; thus, both phages were presumed to be double-stranded DNA phages
(data not shown). In addition, the DNA of pAh1-C was digested by MspI, NcoI, and
HpaII, and its size was estimated to be approximately 55 kb based on the distinct
fragments resulting from NcoI digestion. Likewise, the DNA of pAh6-C was digested by
MspI, SmaI, NcoI, and HpaII, and its size was estimated to be approximately 58 kb based
on the distinct fragments resulting from HpaII digestion (data not shown).
To further characterize pAh1-C and pAh6-C, their structural protein compositions were
analyzed by SDS-PAGE and LC-MS/MS. At least 11 (pAh1-C) and 19 (pAh6-C) distinct
protein bands, with molecular masses ranging from 11 to 105 kDa, were separated. Six
(pAh1-C) and eight (pAh6-C) major protein bands were then subjected to LC-MS/MS for
peptide sequencing (Table 1.3).
The results revealed differences between the structural protein compositions of the two
phages. Although pAh6-C possessed similar structural proteins as other Aeromonas phages
reported, such as the Aeromonas phages Aeh1, 25, and 31, the structural proteins of pAh1-
C were similar to those of phages from species other than Aeromonas (Table 1.3).
1.3.3. Phage stability test
Ethanol treatment resulted in a much greater loss of the activity of both phages
compared to treatment with either chloroform or diethyl ether (Figure 1.3a). Both phages
exhibited similar infection capabilities after incubation at a pH range of 3-11, although
47
pAh6-C was found to be slightly more pH-sensitive than pAh1-C. The optimal stability of
the phages was found at pH 7.0 (Figure 1.3b), and they were relatively heat stable over a
period of 1 h at 20 ~ 25 °C. However, the following reductions in phage activity were
observed for pAh1-C and pAh6-C, respectively: 8.5 ± 0.2% and 19.4 ± 0.6% at 30 °C, 78.7
± 2.3% and 64.7 ± 0.3% at 37 °C, and 97.9 ± 0.1% and 95.3 ± 0.7% at 50 °C. Incubation at
65 °C for 1 h resulted in the complete inactivation of both phages (Figure 1.3c).
1.3.4. Host cell lysis test
The bactericidal effects of pAh1-C and pAh6-C were tested on early-phase A.
hydrophila JUNAH cultures (Figure 1.4). The OD600 values of the uninfected control
culture continued to increase during the incubation period. In contrast, infection with
pAh1-C or pAh6-C inhibited the bacterial growth at MOIs of 0.01, 1, and 100 until 24 h.
However, the bactericidal activity of pAh1-C contrasted with that of pAh6-C. The bacterial
growth of cultures infected with pAh1-C was inhibited until 12 h, regardless of MOI, and
then increased due to the growth of phage-resistant A. hydrophila. In contrast, the
bactericidal activity of pAh6-C varied depending on the MOI. Although the OD600 values
of the cultures infected with pAh6-C increased after 6 h regardless of MOI, the bacterial
growth was inhibited most effectively at an MOI of 1. In addition, the OD600 values of
cultures infected with an MOI of 100 were higher than those of cultures infected with an
MOI of 1, due to the growth of phage-resistant bacteria.
1.3.5. Phage-resistant bacterial variants and pathogenicity in fish
Variant R1, which emerged after exposure to pAh1-C, was also resistant to pAh6-C,
while Variant R2, which emerged after exposure to pAh6-C, was sensitive to pAh1-C.
48
Variant R1 (resistant to both pAh1-C and pAh6-C) and Variant R2 (resistant only to phage
pAh6-C) were used in the pathogenicity tests. The pathogenicities of these variants and
their parent A. hydrophila (JUNAH) strain in loaches are shown in Table 1.2. The phage-
sensitive parent A. hydrophila (JUNAH) caused 100% mortality in the loaches following
intraperitoneal injection with 1.8×107 CFU/fish. All of the fish died within 24 h of
injection, and hemorrhages were observed on their surfaces. The inoculated bacteria were
isolated in pure cultures from the kidneys of the dead fish. In contrast, the variants that
were resistant to pAh1-C and/or pAh6-C were not pathogenic at the same dose (107
CFU/fish) or at a higher dose (108 CFU/fish).
1.3.6. Protective effect of phage administration
The protective effects of intraperitoneal injection and oral administration of the phages
against experimental A. hydrophila (JUNAH) infection are shown in Figure 1.5. In the
first experiment, the fish were challenged with two different doses of bacterial suspension
and were administered two types of phages by IP injection. The fish in the control groups
that were not treated with phages began to die at 1 day post-infection, and the cumulative
mortalities over seven days were 39.17 ± 3.82% (1st trial, 2.6×106 CFU/fish) and 100%
(2nd trial, 2.6×107 CFU/fish). In contrast, the fish treated with phages showed lower
mortality rates; in the 1st trial, no mortality was observed in the groups treated with pAh1-
C or pAh6-C, and in the 2nd trial, the cumulative mortalities were 43.33 ± 2.89% (pAh1-
C) and 16.67 ± 3.82% (pAh6-C) (Figure 1.5a, b).
In the second experiment, the fish were challenged with the bacterial suspension as in
the first experiment but received phage-impregnated feed rather than an intraperitoneal
injection. The fish in the control groups showed mortality rates (1st trial, 38.33 ± 2.50%;
49
2nd trial, 95.83 ± 3.82%) that were similar to those in the first experiment. However, the
fish treated with phages showed lower mortality rates than those of the control group. In
the 1st trial, the cumulative mortality rates were 17.50 ± 2.50% following treatment with
pAh1-C and 11.67 ± 3.82% following treatment with pAh6-C. In the 2nd trial, the
cumulative mortality rates were 46.67 ± 3.82% following treatment with pAh1-C and
26.67 ± 2.89% following treatment with pAh6-C (Figure 1.5c, d). The administration of a
high dose (1010 PFU/fish) of either phage to the experimental fish did not affect their
physical condition or survival during a one-month period of observation. The bacteria were
re-isolated from all of the dead fish except survived fish from the phage-administrated
groups, indicating that the mortalities and protective effects were caused by A. hydrophila
and the phages (pAh1-C and pAh6-C), respectively. In addition, bacteria isolated from the
fish that had received the phage and died were still susceptible to both phages.
1.4. Discussion
In the current study, the indicator host strain JUNAH was determined to be the
causative agent of the mass mortality of cyprinid loaches in a private fish hatchery in
Korea (15). The JUNAH strain has demonstrated resistance to multiple antibiotics and was
therefore able to cause the death of more than 50% of all fish reared on the farm despite
the various antibiotic treatments administered. To discover an effective control agent
against A. hydrophila infection in aquaculture, water samples were collected over a period
of one year for phage isolation.
An increasing incidence of antimicrobial resistance has become a threat to aquaculture
environments (9). The emergence of microbial resistance to multiple drugs due to the
50
liberal and possibly inappropriate use of antibiotics is a serious problem in aquaculture and
causes therapeutic failures (9, 27). Alternatives to conventional antibiotics are urgently
needed for the control of A. hydrophila in aquaculture. Although there have been some
previous reports regarding the isolation of A. hydrophila phages (7, 23, 24, 25, 26), there is
currently a paucity of studies reporting realistic assessments of their therapeutic
applications.
Previously, the therapeutic potential of phages in aquaculture environments was
described (18, 28, 29). In the current study, two phages were isolated from water of the
Han River in Seoul. Although both phages showed a similar host range, they formed
differently shaped plaques and burst sizes, and their gDNAs showed different restriction
endonuclease digestion patterns. In addition, the two phages exhibited different patterns of
bactericidal activity. The method of experimental infection by IP injection was successfully
performed in all experimental groups, in the same manner as previously reported (13, 32).
The challenge trials using various methods such as bath immersion, cohabitation with
infected fish, and oral administration of A. hydrophila-incorporated feed have not resulted
in reduced mortality rates. The in vivo experiments evaluating the protective effects of the
two phages indicated that pAh6-C controlled A. hydrophila infection more effectively than
pAh1-C. Although the phage-resistant bacterial variants were not particularly pathogenic,
the OD600 values of the pAh1-C-infected bacteria gradually increased after 12 h post-
infection.
Wu et al. (41) isolated an A. hydrophila phage, AH1, and performed pathogenicity
testing using loaches. However, these authors infected A. hydrophila with the phage 3 h
before injection of the loaches, and this experiment was performed to indicate that the
pathogenicity of A. hydrophila was eliminated after phage infection and to demonstrate the
51
potential prophylactic use of the phage. Several A. hydrophila phages, such as Aeh1, Aeh2,
PM1, PM2, PM3 and 18, have been reported (7, 23, 24, 25, 26). However, there have been
no reports demonstrating the successful use of phages for the treatment of A. hydrophila
infection or providing evidence of their potential as therapeutic agents. In the current study,
the successful phage treatment of A. hydrophila infection emphasizes the potential of this
therapeutic approach. In addition, pAh1-C and pAh6-C were able to inhibit A. hydrophila
strains from various sources, including three fish-pathogenic isolates (SNUFPC-A6 from
the sailfin molly Poecilia latipinna, SNUFPC-A8 from the cherry salmon Oncorhynchus
masou masou, and JUNAH from the cyprinid loach) and two environmental isolates
(SNUFPC-A20 from a river and SNUFPC-A21 from sewage); these results suggest the
broad application of phages for the treatment of various fish species. Treatment of fish by
IP injection resulted in noticeable protective effects, with increased survival rates observed
for experimental fish compared to the controls. However, treating fish by IP injection in a
fish culture environment is labor-intensive and time-consuming. Therefore, the protective
effect of an oral method of administration was also evaluated for the purpose of identifying
an effective and realistic route of administration on a larger scale, as required on a fish
farm, and this treatment method also improved survival rates considerably.
Despite the improvements in the survival of the phage-treated cyprinid loaches, a
considerable degree of mortality remained. For the successful phage control of A.
hydrophila infection, a combination of both isolated phages may help to increase the
possibility of efficient phage therapy.
52
1.5. References
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Virol. 152: 227-243.
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Spec. Publ. Eur. Aqua. Soc. 10: 11-12.
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5. Cerveny, K.E., et al., 2002. Phage therapy of local and systemic disease caused by
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6. Chhibber, S., Kaur, S., and Kumari, S., 2008. Therapeutic potential of bacteriophage
in treating Klebsiella pneumoniae B5055-mediated lobar pneumonia in mice. J. Med.
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8. Esteve, C., Biosca, E.G., and Amaro, C., 1993. Virulence of Aeromonas hydrophila
and some other bacteria isolated from European eels Anguilla Anguilla reared in fresh
water. Dis. Aquat. Org. 16: 15-20.
9. Giraud, E., et al., 2004. Mechanisms of quinolone resistance and clonal relationship
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53
10. Gutierrez, D., et al., 2010. Isolation and characterization of bacteriophages infecting
Staphylococcus epidermidis. Curr. Microbiol. 61: 601-608.
11. Han, J.E., et al., 2012. First description of the qnrS-like (qnrS5) gene and analysis of
quinolone resistance-determining regions in motile Aeromonas spp. from diseased
fish and water. Res. Microbiol. 163: 73-79.
12. Ishimura, K., et al., 1988. Biochemical and biological properties of motile Aeromonas
isolated from aquatic environments. J. Food Hyg. Soc. Jpn. 29: 313-319.
13. Jeney, G., et al., 2011. Resistance of genetically different common carp, Cyprinus
carpio L., families against experimental bacterial challenge with Aeromonas
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14. Jun, J.W., et al., 2010. Isolation of Aeromonas sobria containing hemolysin gene from
arowana (Scleropages formosus). J. Vet. Clin. 27: 62-66.
15. Jun, J.W., et al., 2010. Occurrence of tetracycline-resistant Aeromonas hydrophila
infection in Korean cyprinid loach (Misgurnus anguillicaudatus). Afr. J. Microbiol.
Res. 4: 849-855.
16. Kim, D.S., Jo, J.Y., and Lee, T.Y., 1994. Induction of triploidy in mud loach
(Misgurnus mizolepis) and its effect on gonad development and growth. Aquaculture
120: 263-270.
17. Kim, H.C., Kim, M.S., and Yu, H.S., 1994. Biological control of vector mosquitoes
by the use of fish predators, Moroco oxycephalus and Misgurnus anguillicaudatus in
the laboratory and semi-field rice paddy. Korean J. Entomol. 24: 269-284.
18. Kim, J.H., et al., 2010. Isolation and identification of bacteriophages infecting ayu
Plecoglossus altivelis altivelis specific Flavobacterium psychrophilum. Vet.
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19. Kim, J.H., et al., 2012. Isolation and characterization of a lytic Myoviridae
bacteriophage PAS-1 with broad infectivity in Aeromonas salmonicida. Curr.
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20. Kumari, S., Harjai, K., and Chhibber, S., 2009. Efficacy of bacteriophage treatment in
murine burn wound infection induced by Klebsiella pneumoniae. J. Microbiol.
Biotechnol. 19: 622-628.
21. Kutter, E., and Sulakvelidze, A., 2005. Bacteriophages: biology and applications.
CRC Press, New York.
22. McGarey, D.J., et al., 1991. The role of motile aeromonads in the fish disease,
ulcerative disease syndrome (UDS). Experientia 47: 441-444.
23. Merino, S., Camprubi, S., and Tomas, J.M., 1992. Characterization of an O-antigen
bacteriophage from Aeromonas hydrophila. Can. J. Microbiol. 38: 235-240.
24. Merino, S., Camprubi, S., and Tomas, J.M., 1990. Identification of the cell surface
receptor for bacteriophage 18 from Aeromonas hydrophila. Res. Microbiol. 141: 173-
180.
25. Merino, S., Camprubi, S., and Tomas, J.M., 1990. Isolation and characterization of
bacteriophage PM2 from Aeromonas hydrophila. FEMS Microbiol. Lett. 68: 239-244.
26. Merino, S., Camprubi, S., and Tomas, J.M., 1990. Isolation and characterization of
bacteriophage PM3 from Aeromonas hydrophila the bacterial receptor for which is
the monopolar flagellum. FEMS Microbiol. Lett. 69: 277-282.
27. Okoh, A.I., and Igbinosa, E.O., 2010. Antibiotic susceptibility profiles of some Vibrio
strains isolated from wastewater final effluents in a rural community of the Eastern
Cape Province of South Africa. BMC Microbiol. 10: 143.
28. Park, S.C., and Nakai, T., 2003. Bacteriophage control of Pseudomonas
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plecoglossicida infection in ayu Plecoglossus altivelis. Dis. Aquat. Org. 53: 33-39.
29. Park, S.C., et al., 2000. Isolation of bacteriophages specific to a fish pathogen,
Pseudomonas plecoglossicida, as a candidate for disease control. Appl. Environ.
Microbiol. 66: 1416-1422.
30. Paterson, W.D., et al., 1969. Isolation and preliminary characterization of some
Aeromonas salmonicida bacteriophages. J. Fish. Res. Can. 26: 629-632.
31. Pathak, S.P., et al., 1988. Seasonal distribution of Aeromonas hydrophila in river
water and isolation from river fish. J. Appl. Bacteriol. 65: 347-352.
32. Reyes-Becerril, M., et al., 2011. Immune response of gilthead seabream (Sparus
aurata) following experimental infection with Aeromonas hydrophila. Fish Shellfish
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33. Rhodes, G., et al., 2000. Distribution of oxytetracycline resistance plasmids between
aeromonads in hospital and aquaculture environments: Implication of Tn1721 in
dissemination of the tetracycline resistance determinant tetA. Appl. Environ.
Microbiol. 66: 3883-3890.
34. Rodgers, C.J., et al., 1981. Quantitative and qualitative studies of Aeromonas
salmonicida bacteriophage. J. Gen. Microbiol. 125: 335-345.
35. Sambrook, J., Fritsch, E.F., and Maniatis, T., 1989. Molecular cloning: a laboratory
manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New
York.
36. Schmidt, A.S., et al., 2001. Incidence, distribution and spread of tetracycline
resistance determinants and integron encoded antibiotic resistance genes among
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5675-5682.
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37. Schmidt, A.S., et al., 2001. Characterization of class 1 integrons associated with R-
plasmids in clinical Aeromonas salmonicida isolates from various geographical areas.
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E., Sulakvelidze, A. (Eds.), Bacteriophages: biology and application. CRC Press,
Boca Raton, pp. 381-436.
39. Verma, V., Harjai, K., and Chhibber, S., 2009. Characterization of a T7-like lytic
bacteriophage of Klebsiella pneumoniae B5055: a potential therapeutic agent. Curr.
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40. Vinodkumar, C.S., Neelagund, Y.F., and Kalsurmath, S., 2005. Bacteriophage in the
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hydrophila, by bacteriophage AH1. Fish Pathol. 15: 271-276.
57
Table 1.1. Bacterial strains used in this study and infectivity of phage pAh1-C and pAh6-C.
Bacterial species (n) Strain Host rangea (EOPsb)
Sourcec pAh1-C pAh6-C
A. hydrophila (5) SNUFPC-A6 + (0.14 ± 0.02) ++ (0.64 ± 0.06) 1
SNUFPC-A8 + (0.34 ± 0.02) + (0.41 ± 0.08) 1
SNUFPC-A20 + (0.11 ± 0.08) + (0.15 ± 0.10) 1
SNUFPC-A21 ++ (0.87 ± 0.03) ++ (0.94 ± 0.02) 1
JUNAH ++ (1.00) ++ (1.00) 2
A. media SNUFPC-A22 - - 1
A. salmonicida ATCC 33658 - - 5
A. sobria Aro - - 6
A. veronii SNUFPC-A17 - - 1
Streptococcus iniae ATCC 29178 - - 5
S. agalactiae ATCC 27956 - - 5
Enterococcus
faecium
CCARM 5192 - - 3
E. faecalis CCARM 5168 - - 3
Vibrio vulnificus ATCC 27562 - - 5
V. parahaemolyticus ATCC 17802 - - 5
V. alginolyticus ATCC 17749 - - 5
Escherichia coli DH10B - - 4 a ++, clear plaque; +, turbid plaque; -, no plaque.
b The EOP (efficiency of plating) values were shown as the mean of observations at three different
occasions.
c 1, Han et al. (11); 2, Jun et al. (15); 3, obtained from the Culture Collection of Antimicrobial
Resistant Microbes in Korea; 4, purchased from Invitrogen; 5, purchased from the American
Type Culture Collection; 6, Jun et al. (14).
58
Table 1.2. Pathogenicities of phage-sensitive parent cells and phage-resistant variants of A.
hydrophila (JUNAH) for loacha.
Strain injected No. of
experiments Dose
(CFU/fish) No. of dead/total fish Mortality (%)
Parent A. hydrophila
(JUNAH)
1st 1.8×106 14/40, 16/40, 15/40 37.50 ± 2.50
2nd 1.8×107 40/40, 40/40, 40/40 100 ± 0
Variant R1 1st 1.4×107 0/40, 0/40, 0/40 0
2nd 1.4×108 0/40, 0/40, 0/40 0
3rd 1.4×109 6/40, 8/40, 6/40 16.67 ± 2.89
Variant R2 1st 1.0×107 0/40, 0/40, 0/40 0
2nd 1.0×108 0/40, 0/40, 0/40 0
3rd 1.0×109 4/40, 6/40, 5/40 12.50 ± 2.50
a Fish were intraperitoneal (IP)-injected with bacteria and were cultivated at 25 ± 1 °C and
observed for 7 days. Variant R1 was resistant to both pAh1-C and pAh6-C; variant R2 was
resistant only to pAh6-C.
59
Table 1.3. SDS-PAGE profiles of pAh1-C and pAh6-C virion and their protein profiles by liquid chromatography-mass spectrometry (LC-MS/MS)
analysis.
MW Protein name [Species] Sequence coverage Accession number
(kDa)
105 e.6 conserved hypothetical protein
[Enterobacteria phage T4] 14.7% NP049742
62 hypothetical protein A2p47
[Lactobacillus phage A2] 13.7% NP680526
49 hypothetical protein EFP gp220
[Enterococcus phage phiEF24C] 33.3% YP001504329
41 gp23
[Mycobacterium phage 244] 26.3% ABD57998
36 bacteriophage protein
[Escherichia coli DEC4D] 35.1% EHV05232
15 gp7
[Mycobacterium phage BarrelRoll] 28.0% AEO94149
60
MW Protein name [Species] Sequence coverage Accession number
(kDa)
105 NrdA-A aerobic NDP reductase large subunit
[Aeromonas phage Aeh1] 56.2% NP943927
95 gp7 base plate wedge initiator
[Aeromonas phage Aeh1] 53.9% NP944089
61 hypothetical protein Aeh1p299
[Aeromonas phage Aeh1] 49.9% NP944177
51 hypothetical protein Aeh1p026
[Aeromonas phage Aeh1] 49.2% NP943904
44 gp6 base plate wedge component
[Aeromonas phage 25] 44.8% YP656367
36 base plate tail tube cap
[Aeromonas phage 31] 41.5% YP238911
18.5 gp68
[Aeromonas phage 31] 58.6% YP238885
17 gp61.1 conserved hypothetical protein
[Aeromonas phage 25] 90.3% YP656265
61
Figure 1.1. Phage plaques formed in double layer agar plates with the indicator host strain, A.
hydrophila JUNAH, and electron micrographs of two phages: (a) pAh1-C (bar = 100 nm) and (b)
pAh6-C (bars = 50 nm).
62
Figure 1.2. One-step growth curve of pAh1-C and pAh6-C. The error bars indicate standard
deviations.
63
Figure 1.3. Stability of pAh1-C (left) and pAh6-C (right) in the presence of various organic solvents
(a), pHs (b), and temperatures (c). To test the stability of these phages in response to the different
factors, optimal conditions such as sterile PBS (a), pH 7 (b), and 4 °C (c) were used as controls. All
values represent the mean of three experiments performed in triplicate on separated occasions, with
error bars representing the standard deviations (SD; n = 3).
64
Figure 1.4. Bactericidal effects of pAh1-C (a) and pAh6-C (b) against A. hydrophila JUNAH. Early
exponential-phase cultures of A. hydrophila JUNAH were co-cultured with pAh1-C (a) and pAh6-C
(b) at MOIs of 0, 0.01, 1, and 100. The results are shown as the mean ± standard deviations from
triplicate experiments.
65
Figure 1.5. Cumulative mortality of different treatment groups following challenge with A.
hydrophila via IP injection. (a) and (b) were treated by IP injection; (c) and (d) were treated by oral
administration of phage-coated feed. control: injected with A. hydrophila (1st, 2.6×106 CFU/fish;
2nd, 2.6×107 CFU/fish) but not treated; pAh1-C: treated with pAh1-C following A. hydrophila
injection; pAh6-C: treated with pAh6-C following A. hydrophila injection. All results are shown as
the mean of triplicate experiments, and error bars represent the SD (n = 3).
66
Chapter II
Bacteriophage Therapy of a Vibrio parahaemolyticus
Infection Caused by a Multiple Antibiotic Resistant O3:K6
Pandemic Clinical Strain
Abstract
Recently isolated Vibrio parahaemolyticus strains have displayed multiple antibiotic
resistance. Alternatives to conventional antibiotics are needed, especially for the multiple-
antibiotic-resistant V. parahaemolyticus pandemic strain. A bacteriophage, designated
pVp-1, that was lytic for V. parahaemolyticus was isolated from the coast of the Yellow
Sea in Korea. The phage showed effective infectivity for multiple-antibiotic-resistant V.
parahaemolyticus and V. vulnificus, including V. parahaemolyticus pandemic strains. The
therapeutic potential of the phage was studied in a mouse model of experimental infection
using a multiple-antibiotic-resistant V. parahaemolyticus pandemic strain. Phage-treated
mice displayed protection from a V. parahaemolyticus infection and survived lethal oral
and intraperitoneal bacterial challenges. This is the first report, to the best of knowledge, of
phage therapy in a mouse model against a multiple-antibiotic-resistant V.
parahaemolyticus pandemic strain infection.
Keywords: Vibrio parahaemolyticus; Bacteriophage; pVp-1; Pandemic strains.
67
2.1. Introduction
Vibrio parahaemolyticus, a gram-negative marine bacterium, is one of the most
important causes of gastroenteritis associated with consumption of raw oysters (7). V.
parahaemolyticus pandemic strains, such as O3:K6, are responsible for the current
pandemics in many countries (14). Emergence of Vibrio species that are resistant to
multiple antibiotics has been recognized as a serious global clinical problem (17). Recently
isolated V. parahaemolyticus pandemic strains have displayed multiple antibiotic resistance,
increasing concerns about possible treatment failure (11).
Theoretically, bacteriophages (phages) can be used to treat infectious diseases both in
humans and animals (2, 4, 5, 13, 20, 24). Phages display an effective bacteriolytic activity
and possess several advantages over other antimicrobial agents, and no serious side effects
of phage therapy have been described thus far (8, 20). Because all isolated V.
parahaemolyticus strains have exhibited resistance to a broad variety of commercial
antibiotics, it has been previously noted that alternatives to conventional antibiotics are
needed (11).
In this study, one lytic Siphoviridae phage, designated as pVp-1 (12) and infects V.
parahaemolyticus pandemic strains, was isolated and characterized. It was aimed to
determine whether this phage could be suitable for therapeutic use in a mouse model of a
multiple-antibiotic-resistant V. parahaemolyticus pandemic strain.
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2.2. Materials and methods
2.2.1. Bacterial strains
V. parahaemolyticus ATCC 33844 was used as the host bacterial strain for phage
isolation and amplification. CRS 09-17 (isolated from a patient with diarrhea; V.
parahaemolyticus new O3:K6 pandemic strain) (11) was used to evaluate its therapeutic
potential.
2.2.2. Electron microscope examination
Phage particles were negatively stained with 2% uranyl acetate, and electron
micrographs were taken using a Zeiss TEM EM902.
2.2.3. One-step growth
The one-step growth curve of pVp-1 was determined according to the method of Verma
et al. (23). Ten microliters of phage suspension was added to 10 ml of the mid-exponential
host bacterial culture (ATCC 33844, 8.0×106 CFU/ml). The mixture was then centrifuged
and the pellet was resuspended in 20 ml of TSB. Samples (100 μl) were taken at 5 min
intervals and subjected to phage titration.
2.2.4. Phage stability
Phage stability tests were conducted as described elsewhere (23), with modifications.
Briefly, phage stability to various conditions such as organic solvents (chloroform, ethanol,
and ether; 25% of total volume), pH (3, 5, 7, 9, and 11), temperature (20, 25, 30, 37, 50,
and 65°C), and UV light (30 cm from the UV-C, 253.7 nm; Sankyo Denki, Japan), was
69
evaluated after 1 h incubation at 25°C (except for the temperature test). After incubation,
the phage titer was estimated by the double-agar-layer method.
2.2.5. Host cell lysis
The bacteriolytic effect of the phage on V. parahaemolyticus CRS 09-17 was observed
by determining viable bacteria counts throughout the incubation period. The phage was
added to the early exponential phase (OD600nm = 0.1; 8.0×106 CFU/ml) of CRS 09-17 at the
indicated multiplicity of infection (MOI), and the change in optical density (OD) was
monitored for 24 h.
2.2.6. Ethics statement
Specific pathogen-free BALB/c female mice (8-weeks-old) were used with the approval
of the Institutional Animal Care and Use Committee, Seoul National University, Seoul,
Republic of Korea (Reg. No. SNU-120602-1). All animal care and experimental protocols
were performed according to the guidelines of the Animal Ethical Committee, Seoul
National University.
2.2.7. Induction of V. parahaemolyticus infection in mice
To determine the 50% lethal dose (LD50), V. parahaemolyticus CRS 09-17 was diluted
with PBS to a range of 2.0×106 to 2.0×108 CFU per mouse in 200 μl and was administered
by either the intraperitoneal (IP) or orogastric route (orally). Five mice were used for each
concentration. The survival rate of mice was recorded until 7 days post-infection. Mice
inoculated with CRS 09-17 were observed for their state of infection based on several
clinical signs, including ruffled fur, hunchback moribund, and partially closed eyes. The
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experiment was replicated three times.
2.2.8. Kinetics of phage in mice
A phage in vivo kinetic assessment was performed as previously described (23), with
several modifications. First, between the two groups, with each group composed of 21
mice, one group was given an IP injection, while the other group was orally given the
phage preparation (2.0×108 PFU/mouse). Second, the two groups (seven mice per group)
were given an IP injection or was oral administered a heat-inactivated (65°C, 2 h) phage
suspension as the negative control. Finally, at appropriate time intervals, four mice (three
test mice and one control mouse) from the IP and oral groups were euthanized, and phage
titers were determined from their organs.
2.2.9. Treatment of bacteremic mice with phage pVp-1
The efficacy of phage therapy was evaluated in two separate experiments using the V.
parahaemolyticus CRS 09-17 infection mouse model. In the first experiment, two groups
of mice (control/treatment; five mice in each group) were challenged by an IP injection of
a LD50 of CRS 09-17. Each mouse was treated with a single IP injection of phage pVp-1
(2.0×108 PFU per mouse) or PBS, 1 h after the bacterial challenge (2.0×107 CFU per
mouse).
In the second experiment, all conditions were similar to those of the first study except
that the bacterial challenge (2.0×107 CFU per mouse) and phage treatment (2.0×108 PFU
per mouse) were administered orally. Both experiments were repeated five times, and the
health condition of the mice was monitored for 72 h.
In an additional study, two groups (five mice per group) were not challenged with
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bacteria and received only phage (2.0×1011 PFU per mouse) by IP and oral routes, as
described previously (22). The state of health of these mice was monitored for 28 days.
2.2.10. Quantitative analysis of V. parahaemolyticus / phage in mouse organs
During the phage treatment experiment described above, three mice from each group
were euthanized at 0, 1, 3, 6, 12, 24, 36, 48, and 72 h post-treatment. As the main target
organs of gastroenteritis, the stomach and intestine were removed and homogenized to
quantify viable bacteria and phage. Bacterial and phage counts were normalized by organ
weight when the organs were halved and processed for histopathological examination. A
selective medium (CHROMagarTM Vibrio containing resistant antibiotics) was used for the
enumeration of V. parahaemolyticus, as described previously (11). This experiment was
repeated three times.
2.2.11. Histopathology of organs
A portion (one-half) of the stomach and intestine was fixed and cut by a standardized
method and placed in tissue cassettes for further processing. Slides of hematoxyline-eosin
stained tissues were prepared and observed for histopathology by microscopic examination.
Histopathology was examined for severity in a blinded manner.
2.2.12. Measuring phage immune response
Mice were immunized using an IP injection of phage (2.0×1010 PFU per mouse) at
intervals of 0, 4, 6, and 8 weeks, as described previously (22). At various times, the sera
from five mice were prepared and indirect enzyme-linked immunosorbent assays were
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performed as previously described (9). Immunoglobulins were detected with goat anti-
mouse Ig (immunoglobulin) M- or IgG-specific antibodies.
2.2.13. Statistical analyses
Statistically significant differences in all of the experiments were determined using
Student’s t-test. The SPSS statistical software package version 16.0 (SPSS, Chicago, IL)
was used for all statistical analyses.
2.3. Results
2.3.1. Characterization of Vibrio phage pVp-1
The Vibrio phage pVp-1 formed small plaques (average diameter, 1 mm) in a lawn of V.
parahaemolyticus ATCC 33844. Based on its morphology, the phage was assigned to the
family Siphoviridae, according to the classification system of Ackermann (1) (Figure 2.1).
Additionally, one-step growth of pVp-1 showed that it had a latent period of approximately
15 min with a burst size of 47 PFU/cell (Figure 2.2).
The pVp-1 was sensitive to organic solvents. After 1 h of incubation in chloroform,
diethylether, and ethanol, phage activity decreased to 37.7%, 33%, and 56.6%, respectively.
However, no effect on phage activity was observed within a pH range of 5-11, and the
activity remained at a high level (94.9%) at pH 3. In addition, the phage was relatively heat
stable over a temperature range of 20-37°C, and no loss in activity was observed, although
phage activity decreased to 3.3% at 50°C and 0% at 65°C. Upon exposure to UV light, a
complete inactivation of pVp-1 at approximately 45 min was observed (Figure 2.9).
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2.3.2. Phage therapeutic application on pandemic clinical strain
The bacteriolytic effect of pVp-1 was tested on an early exponential phase culture of V.
parahaemolyticus CRS 09-17 (Figure 2.3). When the culture was not infected by pVp-1
(control), the OD600 value continued to increase throughout the incubation. In contrast,
bacterial growth induced by pVp-1 was apparently retarded at an MOI of 0.1, 1, and 10
until 24 h. Bacterial growth was properly inhibited at an MOI 1 and 10, whereas the OD600
value at an MOI of 0.1 increased gradually and reached 1.0 after 9 h.
In animal experiments, invalidism and the wellness of the animals were measured using
four criteria: physical condition, survival rate, CFU per gram of target organs (stomach/
intestine), and histopathology of target organs (stomach/intestine). The LD50 of V.
parahaemolyticus CRS 09-17 was examined using IP and oral routes of administration.
The LD50 of the IP and oral routes were between 2.0×106 and 2.0×107 CFU per mouse
(Figure 2.4). The IP and oral mouse infection model was an acute death model; all
mortality occurred within 36 h. After 36 h post-infection, mice that survived entered the
recovery stage.
To examine in vivo kinetics, a pVp-1 kinetic analysis was performed in mice treated by
IP and oral administration (Figure 2.5). In the IP group, a titer of 7.9×105 PFU/ml in blood
during the first hour increased to a titer of 6.4×106 PFU/ml over the next 6 h. However, by
12 h of inoculation, the titer decreased to 2.1×105 PFU/ml. Likewise, pVp-1 titers in the
stomach and intestine were also estimated. In the stomach, a titer of 1.3×104 PFU/g at 1 h
increased to a titer of 3.4×105 PFU/g at 6 h and decreased to 4.5×103 PFU/g at 12 h.
However, in the intestine, a titer of 9.2×104 PFU/g at 1 h increased to a titer of 6.1×106
PFU/g at 12 h and decreased to 4.8×104 PFU/g at 24 h.
In the group receiving the oral administration, a titer of 0 at 1 h in blood increased to a
74
titer of 3.8×106 PFU/ml at 12 h and decreased to 6.7×105 PFU/ml at 24 h. In the stomach, a
titer of 1.1×103 PFU/g at 1 h increased to a titer of 1.2×105 PFU/g at 3 h and decreased to
1.1×104 PFU/g at 6 h. In the intestine, a titer of 2.9×105 PFU/g at 1 h increased to a titer of
5.4×105 PFU/g at 3 h and decreased to 3.4×105 PFU/g at 6 h. There was a gradual fall in
the titer thereafter and after 48 h of inoculation, pVp-1 became undetectable. During the
experiment, all mice were healthy and in normal condition.
To determine whether phage pVp-1 could treat a CRS 09-17 infection, pVp-1 was
administered by IP and oral routes 1 h after a CRS 09-17 challenge (Figure 2.6). After 6 h
of infection, all control (infected but not phage-treated) mice were visibly ill, lethargic, and
scruffy. The control group fatality rate was 56% (IP) and 52% (oral) within 36 h. The
stomachs and intestines of control mice contained high levels of bacteria (stomach, 1.0×104
CFU/g; intestine, 5.3×103 CFU/g) until 12 h post-infection (Figure 2.6). In contrast, phage
treatment resulted in excellent protection in terms of all four criteria. The phage-treated
mice appeared to be only slightly ill and were protected up to 92% (IP) and 84% (oral)
from the lethal infection induced by CRS 09-17 (2.0×107 CFU) after the administration of
a single dose of purified pVp-1 of 2.0×108 PFU (Figure 2.6). The stomach/intestine CFU
and histopathologic features were improved by phage treatment. In the IP treatment group,
a decrease in stomach/intestine CFU was obtained with the increased phage titer at 12 h
post-infection, 1.7×102/3.7×102 CFU/g and 5.7×103/2.3×104 PFU/g (Figure 2.6). In the
oral treatment group, the maximum CFU and PFU values of the stomach/intestine were
observed at experimental onset, and the values gradually decreased (Figure 2.7). In
addition, the histopathologic features demonstrated that the V. parahaemolyticus infection
significantly damaged the intestinal tract in as observed by the H&E stain (Figure 2.8C-D)
but not in the gastric region (data not shown). Severe destruction of the histologic
75
structure of the colon was accompanied by a thinning of the wall, enterohemorrhage, and
loss of crypts in the mucosal layer (Figure 2.8C-D). However, treatment with phage
ameliorated the histological damage in the colon. The histopathological examination of the
colon of the phage-treated mice revealed a significant recovery in the destruction of the
intestinal wall and crypts, hemorrhages and inflammation after both IP and oral treatment
(Figure 2.8E-F). This suggests that phage-treated animals showed strongly reduced
infection severity and could survive a lethal bacterial challenge. In addition, the
administration of a high dose (2.0×1011 PFU/mouse) of pVp-1 alone did not affect the
physical condition or survival during 28 days of observation.
2.3.3. Immune response to phage pVp-1
After the fourth phage induction in a series of phage injections in mice, titers of IgG
and IgM against the phage increased above background levels by 170-fold and 50-fold
respectively (Figure 2.10). No anaphylactic reactions, changes in physical condition or
adverse events were observed during the course of these multiple injections of phage.
2.4. Discussion
Based on the morphological analysis, pVp-1 was classified into the family Siphoviridae,
and it demonstrated a broad host range. This differs from a prior finding that Siphoviridae
phages are generally considered to have restricted host ranges (26). The phage infected
74% (20 / 27) of all multiple antibiotic resistant V. parahaemolyticus strains used in this
study, including the two pandemic strains CRS 09-17 and CRS 09-72. Interestingly, pVp-1
infected the V. parahaemolyticus clinical isolate CRS 09-17 from a patient with diarrhea,
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which represents a multiple-antibiotic-resistant new O3:K6 pandemic strain (tdh+, ORF8+,
toxRS/new+) (11). The results obtained from pVp-1 showed its lytic nature with a latent
period and a large burst size. Full genome sequencing of pVp-1 was identified no
similarity match with lysogenic or phage integrase-related genes (considered as markers of
temperate phages), indicating that it is a novel, newly isolated lytic phage (12). This result
emphasizes the potential of pVp-1 as a therapeutic agent, as described previously by
Gutiérrez et al. (8), who regarded lytic phages as more suitable phages for therapy.
Furthermore, the stability of pVp-1 over a wide range of pH (3-11) and temperature (20-
37°C) clearly indicates that pVp-1 would be highly stable in the body.
Current analyses show that the search for new antibiotics conducted by pharmaceutical
companies is becoming more and more restricted due to the growing costs of conducting
the appropriate trials, low profits, and high risk of the investment, precisely because of the
possibility of a rapid acquisition of resistance to the new drug (6, 16, 21, 25). It was
hypothesized that phage therapy can be useful, especially in epidemics caused by multiple-
antibiotic-resistant pandemic strains. To evaluate the therapeutic potential of pVp-1, a
mouse model of V. parahaemolyticus CRS 09-17 was used. In the cell lysis test of pVp-1,
the growth of CRS 09-17 was apparently inhibited after pVp-1 inoculations at MOIs of 1
and 10, although pVp-1 partially inhibited the bacterial growth at an MOI of 0.1.
Because the route of phage inoculation is important, in vivo phage kinetics were
ascertained following an IP and oral administration of pVp-1. To apply pVp-1 at an MOI of
10 (CRS 09-17, 2.0×107 CFU per mouse), where the maximum effect of cell lysis was
examined, kinetic tests were performed with pVp-1 at 2.0×108 PFU per mouse. Phage titers
in the stomach as well as the intestine indicated that pVp-1 was maintained at higher
concentrations in these two organs and could prevent V. parahaemolyticus infections.
77
These findings suggest that pVp-1 might be efficacious for prophylactic approach. In blood,
pVp-1 reached a high titer within the first hour following IP injection. In contrast, no phage
was detected until 1 h, and it took longer to reach a higher titer, when it was administered
orally. Moreover, the highest titer in each organ was also much higher in the IP route of
administration compared to the oral route of administration. Therefore, it was speculated
that the IP route of administration would be the more suitable route of administration.
Phage treatment trials in the mouse model for CRS 09-17 demonstrated that the
application of pVp-1 can protect from a V. parahaemolyticus infection in all four criteria,
and pVp-1 can be used as a therapeutic agent to reduce the impact of epidemics caused by
multiple-antibiotic-resistant pandemic strains.
While pVp-1 invoked an immune response in mice, the antibodies raised over the
course of repeated injections were not associated with anaphylaxis or other adverse
reactions. These experiments were designed as a model for acute human infections, where
antibiotics are no longer effective and a single course of phage treatment may rescue the
patient. If phages are to be employed repeatedly (e.g., for chronic infections), selection use
and phage display may produce phage variants that are less prone to induce an immune
response (2).
In 2006, the Food and Drug Administration (FDA) approved the use of a commercial
phage cocktail (List-Shield; Intralytix, Inc.) as a biocontrol agent. This is confirmation that
the FDA’s view of phages is that they are safe for human use and opens the doors for phage
commercialization for human applications (10). Despite no reports of significant adverse
reactions during the long history of phage administration in humans, phage therapy still
needs to gain credibility to overcome the regulatory hurdles facing its adoption in
mainstream clinical practice (10). Moreover, it is necessary to establish adequate phage
78
preparation methodologies such as the purification and removal of endotoxins for safety in
phage therapy to prevent anaphylactic responses (3, 15, 18, 19).
2.5. References
1. Ackermann, H.W., 2007. 5500 Phages examined in the electron microscope. Arch.
Virol. 152: 227-243.
2. Biswas, B., et al., 2002. Bacteriophage therapy rescues mice bacteremic from a
clinical isolate of vancomycin-resistant Enterococcus faecium. Infect. Immun. 70:
204-210.
3. Boratynski, J., et al., 2004. Preparation of endotoxin-free bacteriophages. Cell Mol.
Biol. Lett. 9: 253-259.
4. Bruttin, A., and Brűssow, H., 2005. Human volunteers receiving Escherichia coli
phage T4 orally: a safety test of phage therapy. Antimicrob. Agents. Chemother. 49:
2874-2878.
5. Chhibber, S., Kaur, S., and Kumari, S., 2008. Therapeutic potential of bacteriophage
in treating Klebsiella pneumoniae B5055-mediated lobar pneumonia in mice. J. Med.
Microbiol. 57: 1508-1513.
6. Clarke, T., 2003. Drug companies snub antibiotics as pipeline threatens to run dry.
Nature 425: 225.
7. Daniels, N.A., et al., 2000. Vibrio parahaemolyticus infections in the United States,
1973-1998. J. Infect. Dis. 181: 1661-1666.
8. Gutiérrez, D., et al., 2010. Isolation and characterization of bacteriophages infecting
Staphylococcus epidermidis. Curr. Microbiol. 61: 601-608.
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9. Harlow, E., and Lane, D., 1999. Using antibodies, a laboratory manual. Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y.
10. Housby, J.N., and Mann, N.H., 2009. Phage therapy. Drug Discov. Today 14: 536-540.
11. Jun, J.W., et al., 2012. Isolation, molecular characterization, and antibiotic
susceptibility of Vibrio parahaemolyticus in Korean seafood. Foodborne Pathog. Dis.
9: 224-231.
12. Kim, J.H., et al., 2012. Complete genome sequence of a novel marine siphovirus,
pVp-1, infecting Vibrio parahaemolyticus. J. Virol. 86: 7013-7014.
13. Kumari, S., Harjai, K., and Chhibber, S., 2009. Efficacy of bacteriophage treatment in
murine burn wound infection induced by Klebsiella pneumoniae. J. Microbiol.
Biotechnol. 19: 622-628.
14. Matsumoto, C., et al., 2000. Pandemic spread of an O3:K6 clone of Vibrio
parahaemolyticus and emergence of related strains evidenced by arbitrarily primed
PCR and toxRS sequence analyses. J. Clin. Microbiol. 38: 578-585.
15. Merabishvili, M., et al., 2009. Quality-controlled small-scale production of a well-
defined bacteriophage cocktail for use in human clinical trials. PLoS One 4: e4944.
16. Norrby, S.R., et al., 2005. Lack of development of new antimicrobial drugs: a potential
serious threat to public health. Lancet Infect. Dis. 5: 115-119.
17. Okoh, A.I., and Igbinosa, E.O., 2010. Antibiotic susceptibility profiles of some Vibrio
strains isolated from wastewater final effluents in a rural community of the Eastern
Cape Province of South Africa. BMC Microbiol. 10: 143.8.
18. Skurnik, M., Pajunen, M., and Kiljunen, S., 2007. Biotechnological challenges of
phage therapy. Biotechnol. Lett. 29: 995-1003.
19. Skurnik, M., and Strauch, E., 2006. Phage therapy: facts and fiction. Int. J. Med.
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Microbiol. 296: 5-14.
20. Sulakvelidze, A., and Kutter, E., 2005. Bacteriophage therapy in humans. In: Kutter,
E., Sulakvelidze, A., eds. Bacteriophages: biology and applications. Boca Raton, FL:
CRC Press, pp. 381-436.
21. Summers, W.C., 2001. Bacteriophage therapy. Annu. Rev. Microbiol. 55: 437-451.
22. Sunagar, R., Patil, S.A., and Chandrakanth, R.K., 2010. Bacteriophage therapy for
Staphylococcus aureus bacteremia in streptozotocin-induced diabetic mice. Res.
Microbiol. 161: 854-860.16.
23. Verma, V., Harjai, K., and Chhibber, S., 2009. Characterization of a T7-like lytic
bacteriophage of Klebsiella pneumoniae B5055: a potential therapeutic agent. Curr.
Microbiol. 59: 274-281.
24. Vinodkumar, C.S., Neelagund, Y.F., and Kalsurmath, S., 2005. Bacteriophage in the
treatment of experimental septicemic mice from a clinical isolate of multidrug
resistant Klebsiella pneumoniae. J. Commun. Dis. 37: 18-29.11.
25. Wenzel, R.P., 2004. The antibiotic pipeline-challenges, costs, and values. N. Engl. J.
Med. 351: 523-526.
26. Wichels, A., et al., 1998. Bacteriophage diversity in the North Sea. Appl. Environ.
Microbiol. 64: 4128-4133.19.
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Figure 2.1. Electron micrograph of negatively stained phage pVp-1. The bar corresponds to 50 nm.
82
Figure 2.2. One-step growth curve of pVp-1. The error bars indicate standard deviations.
83
Figure 2.3. The bacteriolytic effect of pVp-1 against V. parahaemolyticus CRS 09-17. Early
exponential phase cultures of V. parahaemolyticus CRS 09-17 were co-cultured with pVp-1 at MOIs
of 0, 0.1, 1, and 10. The results are shown as the mean + standard deviations from triplicate
experiments.
84
Figure 2.4. Experimental infection of mouse model. Experimental infection by IP, A or orally, B.
Four test groups were infected (dose volume 0.2 ml) with 2.0×108 CFU per mouse (◆), 2.0×107
CFU per mouse (◇), 2.0×106 CFU per mouse (▲), and 2.0×105 CFU per mouse (△) of V.
parahaemolyticus CRS 09-17. Control (●) group was administered with PBS.
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Figure 2.5. Kinetics of pVp-1 in the mouse model. Phage was injected via IP, A and administered orally, B at 108 PFU/mouse. After 1, 3, 6, 12, 24,
36, and 48 h of phage inoculations, blood, stomach, and intestine were removed and their phage titers were estimated. Titers are presented as the
means of three experiments performed in triplicate, and the error bars represent the SD (n = 3).
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Figure 2.6. Effect of phage treatment on V. parahaemolyticus infection in mice. A, The survival rate
of mice in the control (treated with phosphate buffered saline, PBS) and treated (phage treated)
groups. The LD50 of V. parahaemolyticus CRS 09-17 (2.0×107 CFU/mouse) that was required to
induce an acute death model by way of the IP and oral routes. The phage, pVp-1 (2.0×108
87
PFU/mouse), was applied by IP injection and oral administration after a 1 h CRS 09-17 challenge
because the maximum effect of cell lysis was examined at an MOI of 10. B, CFU per gram of target
organs (stomach/intestine) in the experimental mice through IP infection / IP treatment. C, The PFU
per gram of target organs (stomach/intestine) in the experimental mice through IP infection/ IP
treatment. The bars show the mean, and the error bars show the standard error. Significant
differences (p<0.05) were observed at various time points (shown with asterisks).
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Figure 2.7. CFU and PFU values of the stomach/intestine. Effect of phage treatment on V.
parahaemolyticus infection in mice. A, CFU per gram of target organs (stomach/intestine) in the
experimental mice through oral infection / oral treatment. B, The PFU per gram of target organs
(stomach/intestine) in the experimental mice through oral infection/ oral treatment. In the oral
infection / oral treatment group, the maximum CFU and PFU values of stomach / intestine were
determined at the experimental onset and the values were gradually decreased. The bars show the
mean, and the error bars show the standard error.
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Figure 2.8. Histopathologic features of the intestines of mice infected with V. parahaemolyticus
CRS 09-17 and treated with the phage pVp-1. The micrographs depict the histologic features of the
mice from the experiment. A, A healthy mouse that only received a PBS IP injection. B, A healthy
mouse that only received PBS oral administration. C, A control mouse (IP infection / no phage
treatment). D, A control mouse (oral infection / no phage treatment). Deteriorated crypts are
indicated in C and D. E, A phage-treated mouse (IP infection / IP treatment). F, A phage-treated
mouse (oral infection / oral treatment). The phage-treated mice demonstrated the protected
morphology of the crypt in both the IP and oral treatment groups. Sections were stained with
hematoxylin and eosin and observed at a magnification of ×200.
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Figure 2.9. The sensitivity of pVp-1 to various organic solvents: A, pH: B, temperatures: C, and
exposure to UV light: D. For sensitivity to various factors, optimal conditions included sterile PBS:
A, pH 7: B, 4°C: C, and 0 min: D and acted as a control. All values represent the mean of three
experiments performed in triplicate on different occasions, with error bars representing the standard
deviations (SD; n = 3).
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Figure 2.10. Antibody titers in mice (n = 5) to repeated injections of phage pVp-1. Phage was IP
injected at the indicated time points. The resulting titers of anti-IgG and anti-IgM antibodies are
indicated. Significant differences (p<0.01) were observed at various time points (shown with
asterisks).
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Chapter III
Complete genome sequence of a novel marine siphovirus
pVp-1, infecting Vibrio parahaemolyticus
Abstract
Among the abundant bacteriophages that belong to the order Caudovirales in the ocean,
the genome sequence of marine siphoviruses are poorly investigated in comparison to
myo- or podoviruses. Herein, the complete genome sequence of Vibrio phage pVP-1 was
reported, which belongs to the family Siphoviridae and infects V. parahaemolyticus ATCC
33844.
Keywords: Vibrio parahaemolyticus; Bacteriophage; pVp-1; Siphoviridae.
93
3.1. Introduction
Marine viruses are the most abundant biological entities in the ocean (10), thus making
its genome analysis essential for a better understanding of its enormous genetic diversity
(1). Most reported marine viruses up to date are bacteriophages (phages) that belong to the
order Caudovirales, which is divided into three families: Myoviridae, Podoviridae and
Siphoviridae (10). Among the genome-sequenced marine phages, siphoviruses are
relatively poorly investigated (9) and only two of those including phiHSIC (7) and SIO-2
(1) were studied and reported to infect Vibrio spp.. Herein, the complete genome sequence
of a novel marine siphovirus pVp-1 was reported, which was isolated from the coastal
water of the Yellow sea in Korea and infects V. parahaemolyticus ATCC 33844, which was
isolated from patient with food poisoning.
3.2. Materials and methods
Genomic DNA was extracted as previously described (8), and sequenced using standard
shotgun sequencing reagents and a 454 GS-FLX Titanium Sequencing System (Roche) by
Macrogen in Korea (Approximately 50 × coverage). The full-length genome sequence was
obtained by sequence assembly using the SeqMan II sequence analysis software
(DNASTAR). The putative open reading frames (ORFs) were predicted using Glimmer
3.02 (2) and GeneMark.hmm (6), and putative ORF functions were analyzed by BLASTP
and InterProScan (12). Putative tRNA genes were searched for using tRNAscan-SE (v.
1.21) software (5).
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3.3. Results
The double-stranded and non-redundant DNA genome of pVp-1 was 111,506 bp in
length with a G+C composition of 39.71%. A total of 157 ORFs containing more than 40
amino acid residues and 19 tRNAs (including 1 pseudogene) were identified, suggesting
this as the first marine phage genome in the family Siphoviridae with a large number of
tRNAs capable of infecting V. parahaemolyticus. A number of 48 ORFs showed no
homology to proteins from the GenBank database, while other 69 and 40 ORFs code for
proteins with some homology to known phage and bacteria-related proteins, respectively.
Among the 40 bacteria-related genes in phage pVp-1, 5 ORFs (orf34, orf38, orf79, orf85
and orf97) were highly homologous to Vibrio-related proteins and 35 ORFs shared some
similarities with unrelated bacteria spanning a wide range of phyla. The positions,
directions, sizes, molecular weights, and putative functions of each pVp-1 ORFs are shown
in Table 3.1. The results demonstrate that pVp-1 has a unique genomic composition.
Lysogeny genes responsible for the lysogenic properties of this phage, such as integrase,
were not found in the genome of pVp-1. None of the ORFs showed any similarities with
pathogenecity factors that can cause problems in clinical usage.
Bioinformatic analyses were performed for the assignment of putative functions to 69
phage-related ORFs, and those ORFs were clustered together by at least 3 functional roles
such as DNA metabolism (orf2, orf3, orf4, orf6, orf7, orf12, orf14, orf15, orf16, orf17,
orf18, orf21, orf28, orf32, orf42 and orf52), viral morphogenesis (orf139, orf141, orf143,
orf144, orf148, orf149, orf153, orf155, orf156 and orf157) and lytic properties (orf73,
orf82 and orf83). Interestingly, most ORFs encoding DNA metabolism and viral
morphogenesis genes were clustered together at each ends of the sequenced genome by
95
functional roles, and were similar (≤ 79%) to those of T5 (11) or T5-like phages (3, 4), thus
indicating a close genetic relatedness between pVp-1 and those phages. Genomic
comparison of pVp-1 with the phage T5 revealed that these two phages are highly similar
in gene inventory. The putative ORFs in the pVp-1 genome were predicted using
CoreGenes and had high similarity to the ORFs of the phage T5 (Figure 3.1). Additionally,
according to the ACT comparison results, two parts of the pVp-1 genome was reversed
relative to the order in the T5 genome (Figure 3.2).
In contrast, there were no sequence similarities to marine Vibrio phages belonging to
Siphoviridae (phiHSIC and SIO-2), and high proportions of genes in pVp-1 were not
similar to other sequenced phages or bacteria.
The genome sequence of Vibrio phage pVp-1 was deposited in the GenBank under
accession number JQ340389.
3.4. Discussion
Based on these results, the newly sequenced Vibrio phage pVp-1 could be considered as
a novel T5-like virus, and will help to advance the understanding of the biodiversity of
marine phages belongs to the family Siphoviridae. In addition, a novel virulent phage, pSf-
1, capable of inhibiting V. parahaemolyticus growth indicated the safety needed for phage
therapy against vibriosis.
96
3.5. References
1. Baudoux, A.C., et al., 2012. Genomic and functional analysis of Vibrio phage SIO-2
reveals novel insights into ecology and evolution of marine siphoviruses. Environ.
Microbiol. 14: 2071-2086.
2. Delcher, A.L., et al., 2007. Identifying bacterial genes and endosymbiont DNA with
Glimmer. Bioinformatics 23: 673-679.
3. Hong, J., et al., 2008. Identification of host receptor and receptor-binding module of a
newly sequenced T5-like phage EPS7. FEMS Microbiol. Lett. 289: 202-209.
4. Kim, M.S., and Ryu, S.Y., 2011. Characterization of a T5-like coliphage, SPC35, and
differential development of resistance to SPC35 in Salmonella enterica Serovar
Typhimurium and Escherichia coli. Appl. Environ. Microbiol. 77: 2042-2050.
5. Lowe, T.M., and Eddy, S.R., 1997. tRNAscan-SE: a program for improved detection
of transfer RNA genes in genomic sequence. Nucleic Acids Res. 25: 955-964.
6. Lukashin, A.V., and Borodovsky, M., 1998. GeneMark.hmm: New solutions for gene
finding. Nucleic Acids Res. 26: 1107-1115.
7. Paul, J.H., et al., 2005. Complete genome sequence of φHSIC, a pseudotemperate
marine phage of Listonella pelagia. Appl. Environ. Microbiol. 71: 3311-3320.
8. Sambrook, J., Fritsch, E.F., and Maniatis, T., 1989. Molecular cloning: a laboratory
manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
9. Sullivan, M.B., et al., 2009. The genome and structural proteome of an ocean
siphovirus: a new window into the cyanobacterial ‘mobilome’. Environ. Microbiol.
11: 2935-2951.
10. Suttle, C.A., 2005. Viruses in the sea. Nature 437: 356-361.
97
11. Wang, J., et al., 2005. Complete genome sequence of bacteriophage T5. Virology 332:
45-65.
12. Zdobnov, E.M., and Apweiler, R., 2001. InterProScan – an integration platform for
the signature-recognition methods in InterPro. Bioinformatics 17: 847-848.
98
Table 3.1. Predicted genes and gene products of pVp-1.
Gene Gene product Amino acid
Identity (%) Putative function [organism] (E-value)
Predicted TMH
and signal peptide ORF
No. Range Strand
aa
size
MW
(kD) pI
TMHHM SignalP
1 109-663 + 185 21.1 10.11 31 gp30 DNA ligase
[Acinetobacter phage Acj61] (9.7) 0 N
2 690-1142 - 151 17.0 5.14 47 putative deoxyUTP pyrophosphatase
[Enterobacteria phage T5] (4e-26) 0 N
3 1142-2014 - 291 33.8 4.77 52 flap endonuclease
[Enterobacteria phage T5] (2e-71) 0 N
4 2011-2499 - 163 18.7 8.56 53 D14 protein
[Enterobacteria phage T5] (1e-44) 0 N
5 2492-2785 - 98 11.0 5.72 37 response regulator receiver domain protein
[Bacteroides ovatus SD CC 2a] (3.5) 0 N
6 2778-4652 - 625 71.4 5.72 43 probable exonuclease subunit 2
[Enterobacteria phage T5] (5e-117) 0 N
7 4642-5625 - 328 37.9 5.65 56 putative recombination endonuclease, subunit D12
[Enterobacteria phage T5] (2e-93) 0 N
8 5625-6002 - 126 14.9 4.52 34 ATP-dependent DNA helicase RecG
[Fusobacterium periodonticum ATCC 33693] (3.0) 0 N
9 6049-6852 - 268 29.9 4.74 31 D11 protein
[Enterobacteria phage T5] (8e-28) 0 N
10 6849-7052 - 68 8.2 6.55 55 hypothetical protein T5.125
[Enterobacteria phage T5] (8e-12) 0 N
11 7269 - 7667 - 133 15.3 4.58 27
periplasmic sensor signal transduction histidine
kinase
[Ruegeria sp. R11] (1.0)
0 N
12 7700 - 9055 - 452 51.2 8.34 55 putative ATP-dependent helicase
[Enterobacteria phage T5] (2e-136) 0 N
13 9052 - 9567 - 172 20.5 9.62 38 hypothetical protein
[Enterobacteria phage T5] (3e-25) 0 N
99
14 9557 - 12130 - 858 97.8 5.75 58 DNA polymerase
[Enterobacteria phage T5] (0.0) 0 N
15 12228 - 12983 - 252 29.1 6.39 43 DNA primase
[Enterobacteria phage T5] (3e-51) 0 N
16 13087 - 14487 - 467 52.5 5.04 39 putative replicative DNA helicase
[Enterobacteria phage T5] (7e-97) 0 N
17 14548 - 15312 - 255 27.7 6.05 50 D5 protein
[Enterobacteria phage T5] (4e-52) 0 N
18 15317 - 16093 - 259 28.0 8.72 52 NAD-dependent DNA ligase, subunit B
[Enterobacteria phage T5] (8e-57) 0 N
19 16250 - 16777 - 176 19.8 5.26 32 hypothetical protein
[Pseudomonas phage KPP10] (8e-15) 0 N
20 17008 - 17229 - 74 8.3 5.65 35 hypothetical protein
[Chlamydomonas reinhardtii] (1.6) 0 N
21 17229 - 18197 - 323 36.2 5.14 52 DNA ligase
[Enterobacteria phage T5] (8e-85) 0 N
22 18231 - 18497 - 89 10.2 6.82 28 unnamed protein product
[Tetraodon nigroviridis] (4.3) 0 N
23 18497 - 18862 - 122 13.6 4.56 40
putative bacteriophage protein; putative prohead
protease
[Acinetobacter baumannii AB058] (5.5)
0 N
24 18862 - 19095 - 78 8.8 4.42 0 N
25 19125 - 19430 - 102 11.9 4.74 58 hypothetical protein
[Enterobacteria phage T5] (7e-23) 0 N
26 19414 - 19734 - 107 12.2 9.82 38 hypothetical protein
[Enterobacteria phage T5] (1e-10) 0 N
27 19773 - 20183 - 137 14.7 4.66 36 D3 protein
[Enterobacteria phage T5] (2e-11) 0 N
28 20262 - 21092 - 277 31.8 8.73 35 D2 protein
[Enterobacteria phage T5] (2e-37) 0 N
29 21302 - 21514 - 71 7.8 9.59 42 similar to 60S ribosomal export protein NMD3
[Taeniopygia guttata] (2.3) 0 N
30 21694 – 22626 - 311 35.7 9.32 43 hypothetical protein 0 N
100
[Salmonella phage E1] (2e-33)
31 22587 – 22778 - 64 7.7 10.0 33 phage integrase
[Frankia sp. CcI3] (9.7) 0 N
32 22791 – 25580 - 930 106.8 6.23 46 putative replication origin binding protein
[Enterobacteria phage T5] (0.0) 0 N
33 26246 - 26560 - 105 12.0 6.64 35 hypothetical protein OsI_37948
[Oryza sativa Indica Group] (2.2) 0 N
34 26834 - 28987 + 718 81.0 5.31 58 anaerobic ribonucleoside triphosphate reductase [Vibrio parahaemolyticus 10329] (0.0)
0 N
35 29026 - 29238 + 71 8.3 4.94 32 glutaredoxin 2
[Vibrio sp. MED222] (0.071) 0 N
36 29238 - 30230 + 331 38.5 5.81 64 hypothetical protein ORF027
[Pseudomonas phage PA11] (3e-121) 0 N
37 30230 - 31924 + 565 63.4 5.34 68 hypothetical protein ORF029
[Pseudomonas phage PA11] (0.0) 0 N
38 31926 - 32399 + 158 18.0 7.55 63
anaerobic ribonucleoside-triphosphate reductase
activating protein
[Vibrio parahaemolyticus AQ3810] (7e-50)
0 N
39 32486 - 32884 + 133 15.4 4.19 50 hypothetical protein
[Trichomonas vaginalis G3] (4.4) 0 N
40 32871 – 33314 + 148 17.1 9.05 38 hypothetical protein
[Deftia phage phiW-14] (9e-20) 0 N
41 33295 - 33522 + 76 8.4 7.81 39 Aldo/keto reductase (ISS)
[Ostreococcus tauri] (1.4) 0 N
42 33503 - 33961 + 153 16.9 5.92 46 ribonuclease H
[Enterobacteria phage T5] (4e-31) 0 N
43 33958 - 34158 + 67 8.0 9.90 0 N
44 34206 - 34439 + 78 9.0 5.69 0 N
45 34443 - 34634 + 64 7.7 6.57 36 BcepGomrgp36
[Burkholderia phage BcepGomr] (1.2) 0 N
46 34703 - 34969 + 89 10.1 4.45 53 hypothetical protein
[Enterobacteria phage T5] (0.22) 0 N
47 35038 - 35313 + 92 10.3 10.05 33 hypothetical protein 0 N
101
[Coraliomargarita akajimensis DSM 45221] (9.4)
48 35313 - 35480 + 56 6.5 4.56 36 hypothetical protein
[Acinetobacter phage 133] (0.41) 0 N
49 35473 - 35736 + 88 9.9 11.51 0 N
50 35736 - 35933 + 66 8.0 5.26 36 hypothetical protein
[Shigella phage SP18] (0.014) 0 N
51 36071 - 36316 + 82 9.7 6.28 68 hypothetical protein
[Vibrio phage KVP40] (5.9) 0 N
52 36303 - 37061 + 253 28.1 4.82 32 phage exodeoxyribonuclease
[Escherichia coli TA280] (5e-16) 0 N
53 37137 - 37331 + 65 7.2 10.76 0 N
54 37383 - 37619 + 79 8.6 11.0 2 N
55 37813 - 37968 + 52 5.8 11.74 0 Y
56 39330 - 39698 + 123 14.0 9.22 35 hypothetical protein
[Klebsiella phage KP32] (9.0) 0 N
57 40243 – 40425 + 61 6.8 6.54 0 N
58 40933 – 41337 + 135 15.2 9.12 26 hypothetical protein
[Vibrio phage KVP40] (0.10) 0 N
59 41334 - 41564 + 77 8.8 7.70 32 hypothetical protein
[Trichoplax adhaerens] (3.2) 0 N
60 42004 – 42186 + 61 7.2 10.01 0 N
61 42571 - 42774 + 68 8.8 10.47 48 hypothetical protein
[Aeromonas phage Aeh1] (0.16) 0 N
62 42800 - 43348 + 183 20.6 4.96 41
hypothetical protein
[Staphylococcus carnosus subsp. carnosus TM300]
(8e-26)
0 N
63 43348 - 43980 + 211 23.0 9.33 29 nicotinamide mononucleotide transporter
[Enterobacteria phage EPS7] (1e-10) 6 Y
64 44063 - 44323 + 87 9.8 4.42 35 gp31 protein
[Vibrio phage VP58.5] (0.018) 0 N
65 44316 - 44462 + 49 5.5 4.04 0 N
66 44524 - 44829 + 102 11.4 4.62 38 hypothetical protein SINV_00549
[Solenopsis invicta] (4.3) 0 N
102
67 45031 - 45225 + 65 7.6 9.73 0 N
68 45328 – 45492 + 55 6.4 7.71 1 N
69 45662 – 46285 + 208 23.1 6.22 34 hypothetical protein
[Enterobacteria phage T5] (7e-13) 0 N
70 46653 - 46919 + 89 10.1 9.16 46 hypothetical protein
[Aeromonas phage phiAS4] (3e-12) 0 N
71 46912 – 47283 + 124 14.3 9.00 38 intracellular protein transport protein (UsoA)
[Aspergillus oryzae RIB40] (7.7) 1 Y
72 47280 - 47573 + 98 11.4 8.42 26 PseT.2 conserved hypothetical protein
[Enterobacteria phage T4] (1.2) 0 Y
73 47563 – 48210 + 216 24.3 4.66 30 deoxynucleoside monophosphate kinase
[Enterobacteria phage T5] (2e-10) 0 N
74 48368 - 48703 + 112 12.0 4.27 37 gp71
[Phage phiJL001] (2e-04) 0 N
75 48714 - 51221 + 836 89.8 4.90 31 hypothetical protein
[Vibrio phage ICP2] (2e-15) 0 N
76 51235 - 53820 + 862 93.3 4.58 30 hypothetical protein TU18-25_00220
[Vibrio phage ICP3_2009_A] (0.011) 0 N
77 53859 - 54329 + 157 16.2 4.47 35 hypothetical protein
[Synechococcus elongatus PCC 6301] (9e-05) 0 Y
78 54314 - 54721 + 136 15.7 6.57 37 dipicolinic acid synthetase, A subunit
[Geobacillus sp. Y4.1MC1] (4.9) 1 Y
79 54679 - 55014 + 112 12.3 4.65 31 hypothetical protein VSAK1_25885
[Vibrio shilonii AK1] (5e-07) 0 N
80 55007 - 55165 + 53 5.7 3.92 0 N
81 55162 - 55371 + 70 7.8 7.83 38 CheA signal transduction histidine kinase
[Geobacter lovleyi SZ] (3.5) 0 N
82 55595 - 56266 + 224 25.6 9.00 39 putative holing
[Enterobacteria phage T5] (2e-39) 1 N
83 56268 - 56657 + 130 14.7 9.71 58 gp43 protein
[Enterobacteria phage K1E] (1e-29) 0 N
84 56751 – 57212 + 154 17.9 7.10 30 Metal dependent phosphohydrolase
[Streptococcus phage 8140] (7e-07) 0 N
103
85 57269 – 58309 + 347 39.8 7.77 29 possible DNA binding protein
[Xanthomonas phage Xp15] (1e-13) 0 N
86 58382 - 58684 + 101 12.0 9.65 30 tRNA-splicing endonuclease subunit sen34
[Paracoccidioides brasiliensis Pb01] (3.3) 0 N
87 58704 – 58856 + 51 6.2 7.09 0 N
88 58841 - 59047 + 69 7.7 6.02 37 Metalloreductase
[Cryptococcus gattii WM276] (7.6) 1 Y
89 59038 – 59409 + 124 14.7 5.11 26 acetyl-CoA hydrolase/transferase
[Eubacterium cellulosolvens 6] (0.89) 0 N
90 59402 – 59626 + 75 8.4 9.41 35 conjugation system ATPase, TraG family
[Bacteroides salanitronis DSM 18170] (0.90) 0 N
91 59628 - 59951 + 108 12.1 5.77 33
(dimethylallyl)adenosine tRNA
methylthiotransferase
[Azoarcus sp. EbN1] (0.57)
0 N
92 59935 - 60153 + 73 8.8 10.61 0 N
93 60150 – 60314 + 55 5.9 4.20 1 Y
94 60311 - 60622 + 104 12.0 5.34 30 hypothetical protein PTSG_05611
[Salpingoeca sp. ATCC 50818] (1.6) 1 Y
95 60687 – 61925 + 413 46.2 9.14 37
ABC transporter ATP-binding protein
[Corynebacterium kroppenstedtii DSM 44385]
(4.2)
0 N
96 61985 – 62143 + 53 6.0 9.30 33 unnamed protein product
[Oikopleura dioica] (7.3) 1 N
97 62145 – 63392 + 416 46.3 9.07 24 conserved hypothetical protein
[Vibrio parahaemolyticus 16] (3e-06) 1 Y
98 63456 – 63854 + 133 15.4 9.19 32
periplasmic binding protein
[Bacteroides salanitronis DSM 18170] (2.0)
2 N
99 63842 – 64243 + 134 15.4 6.65 33 ABC transporter related protein
[Clostridium thermocellum DSM 1313] (2.5) 0 N
100 64347 – 65087 + 247 28.4 5.33 32 serine/threonine protein phosphatase
[Enterobacteria phage T5] (8e-23) 0 N
101 65149 – 65349 + 67 7.7 5.18 35 thioesterase superfamily protein 0 N
104
[Desulfococcus oleovorans Hxd3] (9.1)
102 65349 - 65645 + 99 11.3 4.57 38 transcriptional regulator
[Bradyrhizobium japonicum USDA 110] (8.0) 0 N
103 65632 - 66015 + 128 14.9 4.65 31
sodium/potassium-transporting ATPase subunit
alpha-4
[Bos taurus] (5.5)
0 N
104 66056 - 66574 + 173 20.5 4.66 38 RNA helicase, putative
[Trypanosoma brucei gambiense DAL972] (7.1) 0 N
105 66571 – 66945 + 125 14.6 9.17 36 glutamate synthase, NADH/nadph, small subunit
[Paenibacillus polymyxa SC2] (6.2) 0 N
106 66942 – 67148 + 69 8.0 4.79 0 N
107 67301 - 68263 + 321 37.0 6.36 50 conserved hypothetical protein
[Aeromonas phage PX29] (1e-83) 1 N
108 68267 – 68827 + 187 21.1 5.52 61 hypothetical protein
[Aeromonas phage Aeh1] (3e-54) 1 Y
109 68902 - 69183 + 94 11.3 4.44 0 N
110 69183 - 69509 + 109 13.0 9.70 36 conserved hypothetical protein
[Candida albicans WO-1] (6.6) 0 N
111 69547 – 69708 + 54 6.2 6.52 1 N
112 69755 – 70312 + 186 21.0 8.50 24 ORF090
[Staphylococcus phage Twort] (8.7) 0 N
113 70306 - 70455 + 50 5.5 9.10 1 N
114 70457 – 71092 + 212 25.2 9.06 28 beta-lactamase domain protein
[Dethiobacter alkaliphilus AHT 1] (0.011) 0 N
115 71153 - 71485 + 111 13.4 4.19 33 hypothetical protein
[Enterobacteria phage 933W] (3.9) 0 N
116 71463 – 71714 + 84 9.9 5.63 37 argininosuccinate lyase
[Brevibacillus brevis NBRC 100599] (0.90) 0 N
117 71692 - 72018 + 109 12.3 4.29 31
retrotransposon protein, putative, Ty3-gypsy
subclass
[Oryza sativa Japonica Group] (0.83)
0 N
118 72090 – 72470 + 127 14.6 4.29 32 aminopeptidase P
[Reinekea sp. MED297] (4.0) 0 N
105
119 72472 - 72714 + 81 9.1 8.96 25 unnamed protein product
[Oikopleura dioica] (4.4) 0 N
120 72686 - 72910 + 75 8.9 4.47 39 serine/threonine protein kinase
[Methanosarcina barkeri str. Fusaro] (7.3) 0 N
121 72907 – 73146 + 80 9.2 6.54 0 Y
122 73113 - 73247 + 45 5.4 8.40 48
similar to Cytosolic carboxypeptidase 1
(ATP/GTP-binding protein 1)
(Nervous system nuclear protein induced by
axotomy)
[Ciona intestinalis] (9.4)
0 N
123 73514 - 74662 + 383 44.5 8.88 28 hydrolase, haloacid dehalogenase-like family
[Bacillus cereus G9241] (3.8) 0 N
124 74652 – 75011 + 120 14.4 9.40 27 6-phosphofructokinase
[Clostridium difficile QCD-23m63] (2.5) 0 N
125 75238 - 75378 - 47 5.6 10.22 0 N
126 76295 - 76516 - 74 9.0 4.51 32 UDP-galactopyranose mutase
[Leishmania major strain Friedlin] (5.2) 0 N
127 76516 - 76710 - 65 7.8 4.51 30
TRAP family transporter, periplasmic substrate
binding subunit
[Reinekea sp. MED297] (8.7)
0 N
128 76971 - 77288 - 106 12.0 7.86 0 N
129 77316 - 77708 - 131 15.2 5.01 28 chromogranin A (parathyroid secretory protein 1)
[Monodelphis domestica] (1.0) 0 N
130 77805 - 78125 - 107 12.3 3.86 25 Transketolase
[Salinispora arenicola CNS-205] (3.4) 0 N
131 78192 -
78314 - 41 4.7 5.96 0 N
132 79248 - 79718 + 157 16.8 5.07 38 A2 protein
[Enterobacteria phage T5] (5e-09) 0 N
133 79815 - 80186 + 124 13.8 9.37 25 DEAD/DEAH box helicase, putative
[Arabidopsis thaliana] (2.9) 0 N
134 80288 - 81943 + 552 61.8 6.14 31 A1
[Enterobacteria phage T5] (2e-50) 0 N
106
135 82024 - 82662 + 213 24.5 4.57 41 5'-deoxyribonucleotidase
[Enterobacteria phage T5] (2e-27) 0 N
136 82810 - 83478 - 223 25.5 8.61 28 hypothetical protein
[Enterobacteria phage T5] (8e-08) 0 N
137 83490 - 85427 - 646 72.6 9.67 30 hypothetical protein EUBDOL_02205
[Eubacterium dolichum DSM 3991] (2.2) 0 N
138 85549 - 86019 + 157 17.6 5.12 49 hypothetical protein
[Enterobacteria phage T5] (8e-27) 0 N
139 86019 - 87338 + 440 50.8 5.04 59 terminase large subunit
[Enterobacteria phage T5] (9e-144) 0 N
140 87420 - 87890 + 157 18.1 7.75 51 hypothetical protein
[Enterobacteria phage T5] (5e-31) 0 N
141 87893- 89119 + 409 46.1 5.30 51 portal protein
[Enterobacteria phage T5] (1e-123) 0 N
142 89109 - 89354 + 82 9.6 9.24 30 hypothetical protein
[Pyrobaculum calidifontis JCM 11548] (9.0) 0 N
143 89341 - 89946 + 202 22.3 4.78 54 putative prohead protease
[Enterobacteria phage T5] (1e-53) 0 N
144 89954 - 91351 + 466 51.6 5.46 60 major head protein precursor
[Enterobacteria phage T5] (2e-142) 0 N
145 91412 - 91954 + 181 20.3 5.24 22 hypothetical protein
[Enterobacteria phage EPS7] (7e-04) 0 N
146 91954 - 92700 + 249 28.3 9.95 45 hypothetical protein
[Enterobacteria phage T5] (1e-52) 0 N
147 92697 - 93182 + 162 18.9 4.86 38 hypothetical protein
[Enterobacteria phage T5] (1e-26) 0 N
148 93197 - 94612 + 472 51.7 4.68 45 major tail protein
[Enterobacteria phage T5] (2e-104) 0 N
149 94618 - 95502 + 295 32.7 6.14 29 minor tail protein
[Enterobacteria phage T5] (8e-17) 0 N
150 95499 - 95909 + 137 15.6 4.72 33 hypothetical protein
[Enterobacteria phage T5] (5e-16) 0 N
151 95920 - 96312 + 131 15.2 9.06 35 hypothetical protein 0 N
107
[Enterobacteria phage T5] (9e-14)
152 96394 - 97152 + 253 27.6 5.62 27 6-phosphogluconate dehydrogenase
[Vibrio salmonicida LFI1238] (3.1) 0 N
153 97168 -
100239 + 1024 111.2 6.14 39
pore-forming tail tip protein pb2
[Enterobacteria phage T5] (3e-71) 0 N
154 100404 -
101021 + 206 23.1 4.85 54
hypothetical protein
[Enterobacteria phage T5] (1e-55) 0 N
155 101018 -
103861 + 948 106.9 5.12 46
structural tail protein
[Enterobacteria phage T5] (0.0) 0 N
156 103858 -
106944 + 1029 111.1 6.01 46
tail protein Pb4
[Enterobacteria phage T5] (5e-131) 0 N
157 106947 -
107729 + 261 28.0 9.44 22
putative phage tail protein
[Enterobacteria phage EPS7] (5e-05) 0 N
108
Figure 3.1. Genome map of phage pVp-1. Hypothetical functions of encoded proteins were determined through comparison of amino acid
sequences to the non-redundant databank using BLASTP. The + and – stranded ORFs were colored as grey and white, respectively. The CoreGenes
between phage pVp-1 and phage T5 were colored as green.
109
Figure 3.2. Genome comparison of phage pVp-1 to its relative phage (T5) using Artemis
Comparison Tool (ACT). Translated BLAST (tblastx, score cutoff: 40) was used to align translated
genome sequences of phages. The blue lines represent the reverse and forward matches, and color
intensity is proportional to the sequence homology. Nucleotide base-pairs were indicated between
grey lines for each phage genomes.
110
GENERAL CONCLUSION
Recently, there has been an increasing appreciation of their role as waterborne pathogens
of fish and humans. There has been an increasing incidence of antimicrobial resistance
among Aeromonas sp. isolated from aquaculture environments. Multiple-antibiotic-
resistant A. hydrophila exists in aquaculture systems and contributes to the high rate of
mortality within the fish industry in Korea. Although the majority of the loach population
in Korea is cultured and A. hydrophila is one of the main causes of mass mortality in these
fish, no effective method has been proposed for the control of A. hydrophila infection in
aquaculture, except for the application of additional antibiotics. In the first step, to
investigate methods to control the mass mortality of cyprinid loaches (Misgurnus
anguillicaudatus) caused by multiple-antibiotic-resistant Aeromonas hydrophila on a
private fish farm in Korea, bacteriophages (phages), designated pAh1-C and pAh6-C, were
isolated from the Han River in Seoul. The two isolated phages were morphologically
classified as Myoviridae and showed similar infection patterns for A. hydrophila isolates.
The phages proved to be efficient in the inhibition of bacterial growth, as demonstrated by
their in vitro bactericidal effects. Additionally, a single administration of either phage to
cyprinid loaches resulted in noticeable protective effects, with increased survival rates
against A. hydrophila infection.
Vibrio parahaemolyticus is one of the most important causes of gastroenteritis. Although
raw oysters have such high densities of V. parahaemolyticus that the consumption of raw
oysters is known to cause illness in humans, almost all Koreans prefer raw oysters to
already cooked oysters because of their fresh taste and high nutritional value. V.
parahaemolyticus pandemic strains, such as O3:K6, are responsible for the current
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pandemics in many countries. Emergence of Vibrio species that are resistant to multiple
antibiotics has been recognized as a serious global clinical problem. Recently isolated V.
parahaemolyticus pandemic strains have displayed multiple antibiotic resistance,
increasing concerns about possible treatment failure. Alternatives to conventional
antibiotics are needed, especially for the multiple-antibiotic-resistant V. parahaemolyticus
pandemic strain. In the second step, a bacteriophage, designated pVp-1, that was lytic for V.
parahaemolyticus was isolated from the coast of the Yellow Sea in Korea. The phage
showed effective infectivity for multiple-antibiotic-resistant V. parahaemolyticus and V.
vulnificus, including V. parahaemolyticus pandemic strains. The therapeutic potential of the
phage was studied in a mouse model of experimental infection using a multiple-antibiotic-
resistant V. parahaemolyticus pandemic strain. Phage-treated mice displayed protection
from a V. parahaemolyticus infection and survived lethal oral and intraperitoneal bacterial
challenges.
In the third step, the complete genome sequence of a novel marine siphovirus pVp-1 was
reported, which was isolated from the coastal water of the Yellow sea in Korea and infects
V. parahaemolyticus. Genomic comparison of pVp-1 with the phage T5 revealed that these
two phages are highly similar in gene inventory.
Based on these results, it is clear that phages can be considered as altenative therapeutic
or prophylactic candidates against bacterial infections in humans as well as fish. The use of
biocontrol method using phages seems to be a promising alternative to conventional
antibiotics.
112
국
박테리 파아지 유전체연구 및 Aeromonas hydrophila
Vibrio parahaemolyticus에 대한 박테리 파아지 치료법
2011-31100 진우
공 보건 공
울 과 원
Aeromonas spp.는 상 인 경에 존재 는 주요 균 , 근 어
뿐만 아니라 인간에게도 해를 는 인 병원균 주목 고 있다.
Aeromonas hydrophila는 어 에 질병 는 운동 aeromonad 일종이며
질병 생시 높 폐사를 야 다. 근 양식 경에 분리 Aeromonas sp.
에 항생 내 생 도 증가가 보고 고 있다. 여러 종 항생 에
내 획득 A. hydrophila는 실 양식 경에 존재 고 있 며 국 어업에
많 폐사를 일 키고 있다. 국내산 미꾸라지 부분 양식산이며 A.
hydrophila는 미꾸라지 폐사 주요 원인 알 있지만, 양식산업에 A.
hydrophila 감염증에 책 항생 여외에는 그 어떤 효과 인 책
도 없는 실 이다. 국내 어 양식장에 생 다 항생 에 내 보
이는 A. hydrophila 감염에 미꾸라지 폐사를 막 안 연구
여, 강에 pAh1-C pAh6-C라고 명명 리 아지 (이후 아지)
를 분리 다. 개 아지는 태 Myoviridae 분 었고 A.
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hydrophila 균주들에 사 감염 양상 나타내었다. 개 아지는 55 kb
(pAh1-C) 58 kb (pAh6-C) 크 double-strand DNA를 보 고 있었 며, 그
아지 DNA는 효소 이용 분 에 다른 보 다.
아지는 모 어 에 병원 는 A. hydrophila에 해 효과 인 균
억 능 갖고 있는 것 드러났다. 아지 latent period는 략 30
분 (pAh1-C)과 20 분 (pAh6-C) 찰 었고 burst size는 60 (pAh1-C)과 10
(pAh6-C) 었다. 생체외 실험실 상에 찰 균 억 능
에 드러난 같이, 본 아지들 균 장 효과 억 는 것
명 었다. 미꾸라지를 상 일회 아지 여는 A.
hydrophila 감염증에 해 목 만 어 효과를 나타내며 생존 이 크게 향
상 었다. 이러 연구 결과는 pAh1-C pAh6-C, 이 아지가 어 에 A.
hydrophila 감염증에 료 충분 가능 갖고 있 증명 고
있다.
Vibrio parahaemolyticus는 장 질 는 주요 원인체이다. 본 감염
증 불 게 조리 해산 , 특히 굴 생식과 게 어 있다.
계 V. parahaemolyticus는 주요 감염증 원인 알 있다. 동아시
아인들, 특히 국인과 일본인 독특 식습 가지고 있다. 국인들 다
양 종 어 어 를 생식 다. 생굴 다량 V. parahaemolyticus를 함
고 있 며 생굴 취는 질병 생 험 이 있 에도 불구 고, 부분
국인들 그 신 풍미 높 양소를 즐 여 조리 굴보다
생굴 다. O3:K6 같 V. parahaemolyticus pandemic strain 계 여
114
러 국가에 규모 식 독 원인균 알 있다. 다 항생 에 내
보 Vibrio 균 출 심각 범 계 인식 어 다. 근
분리 V. parahaemolyticus pandemic strain들 다 항생 에 내 나타내었
며 이는 감염 었 경우 료 실 가능 에 우 를 증폭시키고 있
다. 이에, 다 항생 내 V. parahaemolyticus pandemic strain에 항생
체 법이 실히 요구 고 있다. V. parahaemolyticus에 감염 며 pVp-1
명명 아지가 황해 연안에 분리 었다. 아지는 V. parahaemolyticus
pandemic strain 포함 여, 다 항생 내 V. parahaemolyticus V.
vulnificus에 효과 인 감염 나타내었다. 다 항생 내 V.
parahaemolyticus pandemic strain 이용 마우스 감염 실험 모델 용 여 본
아지 료 가능 에 여 검증 다. 아지를 여 마우스
는 V. parahaemolyticus 감염증에 여 충분 어 보 며, 식이 복강
주사를 통 사량에 달 는 균 감염에 도 생존 나타내었다. 본 연구
는 다 항생 내 V. parahaemolyticus pandemic strain에 아지 료
보고이다.
V. parahaemolyticus에 감염 는 신규 해양 siphovirus pVp-1가 황해 연안에
분리 었고, 이 체가 보고 었다. pVp-1 double-strand DNA 체는
111,506 bp, 39.71% G + C 함량 분 었다. pVp-1 체 분 에 는
pVp-1이 T5 아지 높 체 사도를 갖고 있다는 것이 증명 었고,
이는 pVp-1과 T5 사이 체 시사 는 것이었다. pVp-1
과 T5를 상 는 체 사 분 결과에 는 이 아지가
115
체 구 에 있어 높 사 보 고 있 이 시 었다.
이러 결과를 탕 , 항생 내 A. hydrophila에 감염 는 Aeromonas
아지가 양식 경에 Aeromonas 감염증에 료
충분 가능 보 고 있 이 인 었다. V. parahaemolyticus
CRS 09-17 균 이용 마우스 상 아지 료 실험 pVp-1 여가 V.
parahaemolyticus 감염증 부 어를 가능 게 며, 다 항생 내
pandemic strain에 해 야 는 규모 염병 해를 소 는데 료
사용 있다는 것이 증명 었다.
Key words: Aeromonas hydrophila, 리 아지 ( 아지), Vibrio
parahaemolyticus, 다 항생 내 pandemic strain, 료 .
Student number: 2011-31100
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List of published articles
2013
1. Jin Woo Jun, Tae-Hoon Shin, Ji Hyung Kim, Sang Phil Shin, Jee Eun Han, Gang
Joon Heo, Mahanama De Zoysa, Gee Wook Shin, Ji Young Chai, Se Chang Park
(2013). Bacteriophage therapy of a Vibrio parahaemolyticus infection caused by a
multiple antibiotic resistant O3:K6 pandemic clinical strain. J. Infect. Dis. In Press.
2. Jin Woo Jun, Ji Hyung Kim, Sang Phil Shin, Jee Eun Han, Ji Young Chai, Se Chang
Park (2013). Characterization and complete genome sequence of the Shigella
bacteriophage pSf-1. Res. Microbiol. doi. 10. 1016/j. resmic.2013.08.007.
3. Jin Woo Jun, Ji Hyung Kim, Sang Phil Shin, Jee Eun Han, Ji Young Chai, Se Chang
Park (2013). Protective effects of the Aeromonas phages pAh1-C and pAh6-C against
mass mortality of the cyprinid loach (Misgurnus anguillicaudatus) caused by
Aeromonas hydrophila. Aquaculture 416-417:289-295.
4. Jin Woo Jun, Ji Hyung Kim, Casiano H. Choresca Jr., Sang Phil Shin, Jee Eun Han,
Se Chang Park (2013). Draft Genome Sequence of Vibrio parahaemolyticus
SNUVpS-1 Isolated from Korean Seafood. Genome Announcement 1(1): e00132-12.
5. Sang Phil Shin, Mun Sup Kim, Sung Hee Cho, Ji Hyung Kim, Casiano H. Choresca Jr.,
Jee Eun Han, Jin Woo Jun, Se Chang Park (2013). Antimicrobial effect of
hypochlorous acid on pathogenic microorganisms. J. Prev. Vet. Med. 37(1):49-52.
6. Sang Phil Shin, Ji Hyung Kim, Casiano H. Choresca Jr., Jee Eun Han, Jin Woo Jun,
Se Chang Park (2013). Molecular identification and phylogenetic characterization of
Thelohanellus kitauei. Acta Vet. Hung. 61(1):30-35.
117
7. Jee Eun Han, Ji Hyung Kim, Tristan Renault, Casiano H. Choresca Jr., Sang Phil Shin,
Jin Woo Jun, Se Chang Park (2013). Identifying the Viral Genes Encoding Envelope
Glycoprotein for Differentiation of Cyprinid herpesvirus 3 Isolates. Viruses-Basel
5(2):568-576.
8. Jee Eun Han, Ji Hyung Kim, Casiano H. Choresca Jr., Sang Phil Shin, Jin Woo Jun,
Se Chang Park (2013). Draft Genome Sequence of a Clinical Isolate, Aeromonas
hydrophila SNUFPC-A8, from a Moribund Cherry Salmon (Oncorhynchus masou
masou). Genome Announcement 1(1):e00133-12.
9. Jee Eun Han, Ji Hyung Kim, Casiano H. Choresca Jr., Sang Phil Shin, Jin Woo Jun,
Se Chang Park (2013). Body extract of tail amputated zebrafish promotes culturing of
primary fin cells from glass catfish. Afr. J. Biotechnol. 12(12):1449-1451.
10. Ji Hyung Kim, Casiano H. Choresca Jr., Sang Phil Shin, Jee Eun Han, Jin Woo Jun,
Se Chang Park (2013). Biological Control of Aeromonas salmonicida subsp.
salmonicida Infection in Rainbow Trout (Oncorhynchus mykiss) Using Aeromonas
Phage PAS-1. Transbound. Emerg. Dis. doi: 10. 1111/tbed. 12088.
11. Jee Eun Han, Ji Hyung Kim, Sun Young Hwang, Casiano H. Choresca Jr., Sang Phil
Shin, Jin Woo Jun, Ji Young Chai, Yong Ho Park, Se Chang Park (2013). Isolation
and characterization of a Myoviridae bacteriophage against Staphylococcus aureus
isolated from dairy cows with mastitis. Res. Vet. Sci. 95:758-763.
12. Jee Eun Han, Ji Hyung Kim, Sang Phil Shin, Jin Woo Jun, Ji Young Chai, Se Chang
Park (2013). Draft genome sequence of Aeromonas salmonicida subsp. achromogenes
AS03, an atypical strain isolated from Crucian Carp (Carassius carassius) in the
Republic of Korea. Genome Announcement 1(5): e00791-13.
13. Jee Eun Han, Ji Hyung Kim, Casiano Choresca Jr., Sang Phil Shin, Jin Woo Jun, Se
118
Chang Park (2013). Sequence-based genotyping methods to assess the genetic
diversity of Riemerella anatipestifer isolates from ducklings with tremor. New
Microbiol. 36(4):395-404.
14. Jee Eun Han, Sun Young Hwang, Ji Hyung Kim, Sang Phil Shin, Jin Woo Jun, Ji
Young Chai, Yong Ho Park, Se Chang Park (2013). Methicillin resistant coagulase-
negative staphylococci isolated from ducks. Acta Vet. Scand. In Press.
15. Sang Phil Shin, Sang Yoon Han, Casiano H. Choresca Jr., Jee Eun Han, Jin Woo Jun,
Ji Hyung Kim, Se Chang Park (2013). Expression and characterization of cathepsin L-
like cysteine protease from Philasterides dicentrarchi. Parasitol. Int. In Press.
16. Sang Phil Shin, Van Giap Nguyen, Jae Mook Jeong, Jin Woo Jun, Ji Hyung Kim,
Casiano H. Choresca Jr., Jee Eun Han, Gun Wook Baeck, Se Chang Park (2013). The
phylogenetic study on Thelohanellus species (Myxosporea) in relation to host
specificity and infection site tropism. Mol. Phylogenet. Evol. In Press.
2012
1. Jin Woo Jun, Ji Hyung Kim, Casiano Choresca Jr., Sang Phil Shin, Se Chang Park
(2012) Isolation, molecular characterization and antibiotic susceptibility of Vibrio
parahaemolyticus in Korean seafood. Foodborne Pathog. Dis. 9(3):224-231.
2. Ji Hyung Kim, Jin Woo Jun, Casiano H. Choresca, Sang Phil Shin, Jee Eun Han, Se
Chang Park (2012) Complete genome sequence of a novel marine siphovirus pVp-1,
infecting Vibrio parahaemolyticus. J. Virol. 86(12):7013-7014.
3. Ji Hyung Kim, Hye Kwon Kim, Van Giap Nguyen, Bong Kyun Park, Casiano H.
Choresca Jr., Sang Phil Shin, Jee Eun Han, Jin Woo Jun, Se Chang Park (2012)
119
Genomic sequence of infectious hypodermal and hematopoietic necrosis virus
(IHHNV) KLV-2010-01 originating from the first Korean outbreak in cultured
Litopenaeus vannamei. Arch. Virol. 157:369-373.
4. Ji Hyung Kim, Jee Soo Son, Casiano H. Choresca, Sang Phil Shin, Jee Eun Han, Jin
Woo Jun, Do Hyung Kang, Chulhong Oh, Soo Jin Heo, Se Chang Park (2012)
Complete Genome sequence of bacteriophage phiAS7, a T7-like virus that infects
Aeromonas salmonicida subsp. salmonicida. J. Virol. 86(5):2894.
5. Ji Hyung Kim, Jee Soo Son, Yoon Jae Choi, Casiano H. Choresca, Sang Phil Shin, Jee
Eun Han, Jin Woo Jun, Se Chang Park (2012) Complete genomic sequence of a T4-
like bacteriophage phiAS4 infecting Aeromonas salmonicida subsp. salmonicida. Arch.
Virol. 157:391-395.
6. Ji Hyung Kim, Jee Soo Son, Yoon Jae Choi, Casiano H. Choresca, Sang Phil Shin, Jee
Eun Han, Jin Woo Jun, Do Hyung Kang, Chulhong Oh, Soo Jin Heo, Se Chang Park
(2012) Isolation and characterization of a lytic Myoviridae bacteriophage PAS-1 with
broad infectivity in Aeromonas salmonicida. Curr. Microbiol. 64:418–426.
7. Ji Hyung Kim, Jee Soo Son, Yoon Jae Choi, Casiano H. Choresca, Sang Phil Shin, Jee
Eun Han, Jin Woo Jun, Se Chang Park (2012) Complete genome sequence and
characterization of a broad-host range T4-like bacteriophage phiAS5 infecting
Aeromonas salmonicida subsp. salmonicida. Vet. Microbiol. 157:164–171.
8. Ji Hyung Kim, Chul Hong Oh, Casiano Choresca Jr., Sang Phil Shin, Jee Eun Han, Jin
Woo Jun, Soo Jin Heo, Do Hyung Kang, Se Chang Park (2012) Complete genome
sequence of bacteriophage phiAC-1 infecting Acinetobacter soli KZ-1. J. Virol.
86(23):13131.
120
9. Sang Phil Shin, Hyo Jin Yang, Ji Hyung Kim, Casiano H. Choresca Jr. Jee Eun Han,
Jin Woo Jun, Sang Yoon Han, Se Chang Park (2012). Rapid detection and isolation
of Salmonella sp. from amphibians and reptiles. Afr. J. Biotechnol. 11(24):682-686.
10. Jee Eun Han, Ji Hyung Kim, Casiano Choresca Jr., Sang Phil Shin, Jin Woo Jun, Ji
Young Chai, Sang Yoon Han, Se Chang Park (2012) First description of the qnrS-like
(qnrS5) gene and analysis of quinolone resistance-determining regions in motile
Aeromonas spp. from diseased fish and water. Res. Microbiol. 163:73–79.
11. Jee Eun Han, Ji Hyung Kim, Casiano Choresca Jr., Sang Phil Shin, Jin Woo Jun, Ji
Young Chai, Sang Yoon Han, Se Chang Park (2012) Prevalence of tet gene and
complete genome sequencing of tet gene-encoded plasmid (pAHH01) isolated from
Aeromonas species in South Korea. J. Appl. Microbiol. 112:631-638.
12. Jee Eun Han, Ji Hyung Kim, Casiano Choresca Jr., Sang Phil Shin, Jin Woo Jun, Ji
Young Chai, Sang Yoon Han, Se Chang Park (2012) A small IncQ-type plasmid
carrying the quinolone resistance (qnrS2) gene from Aeromonas hydrophila. Lett.
Appl. Microbiol. 54:374-376.
13. Casiano Choresca Jr., Casiano H. Choresca Jr.1, Jung Taek Kang, Jee Eun Han, Ji
Hyung Kim, Sang Phil Shin, Jin Woo Jun, Byeong Chun Lee, Se Chang Park (2012)
Effect of storage media and time on fin explants culture in the goldfish, Carassius
auratus. Afr. J. Biotechnol. 11(24):6599-6602.
14. Jee Eun Han, Ji Hyung Kim, Casiano Choresca Jr., Sang Phil Shin, Jin Woo Jun, Ji
Young Chai, Se Chang Park (2012) First description CoIE-type plasmid in Aeromonas
spp. carrying quinolone resistance (qnrS2) gene. Lett. Appl. Microbiol. 55:290-294.
121
2011
1. Jin Woo Jun, Ji Hyung Kim, Casiano H. Choresca Jr., Sang Phil Shin, Jee Eun Han,
Dal Sang Jeong, Se Chang Park (2011) Isolation and molecular detection of
Plesiomonas shigelloides containing tetA gene from Asian arowana (Scleropages
formosus) in a Korean aquarium. Afr. J. Microbiol. Res. 5(28):5019-5021.
2. Dennis K. Gomez, Seong Joon Joh, Hwan Jang, Sang Phil Shin, Casiano H. Choresca
Jr., Jee Eun Han, Ji Hyung Kim, Jin Woo Jun, Se Chang Park (2011). Detection of
koi herpesvirus (KHV) from koi (Cyprinus carpio koi) broodstock in South Korea.
Aquaculture 311:42-47.
3. Ji Hyung Kim, Casiano H. Choresca Jr., Sang Phil Shin, Jee Eun Han, Jin Woo Jun,
Se Chang Park (2011). Occurrence and antibiotic resistance of Vibrio vulnificus in
seafood and environmental waters in Korea. J. Food Saf. 31:518-524.
4. Ji Hyung Kim, Sun Young Hwang, Jee Soo Son, Jee Eun Han, Jin Woo Jun, Sang
Phil Shin, Casiano H. Choresca Jr., Yun Jaie Choi, Yong Ho Park, Se Chang Park
(2011). Molecular characterization of tetracycline- and quinolone-resistant Aeromonas
salmonicida isolated in Korea. J. Vet. Sci. 12(1):41-48.
5. Ji Hyung Kim, Casiano H. Choresca Jr., Sang Phil Shin, Jee Eun Han, Jin Woo Jun,
Sang Yoon Han, Se Chang Park (2011). Detection of infectious hypodermal and
hematopoietic necrosis virus (IHHNV) in Litopenaeus vannamei shrimp cultured in
South Korea. Aquaculture 313:161-164.
6. Casiano H. Choresca Jr., Dennis K. Gomez, Sang Phil Shin, Ji Hyung Kim, Jee Eun
Han, Jin Woo Jun, Se Chang Park (2011). Molecular detection of Edwardsiella tarda
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with gyrB gene isolated from pirarucu, Arapaima gigas which is exhibited in an
indoor private commercial aquarium. Afr. J. Biotechnol. 10(5):848-850.
7. Sang Phil Shin, Dennis K. Gomez, Ji Hyung Kim, Casiano H. Choresca Jr., Jee Eun
Han, Jin Woo Jun, Se Chang Park (2011). Detection and genetic analysis of
aquabirnaviruses in subclinically infected aquarium fish. J. Vet. Diagn. Invest. 23:325-
329.
8. Sang Phil Shin, Jee Eun Han, Dennis K. Gomez, Ji Hyung Kim, Casiano H. Choresca
Jr., Jin Woo Jun, Se Chang Park (2011). Identification of scuticociliate Philasterides
dicentrarchi from indo-pacific seahorses Hippocampus kuda. Afr. J. Microbiol. Res.
5(7):738-741.
9. Sang Phil Shin, Hyang Jee, Jee Eun Han, Ji Hyung Kim, Casiano H. Choresca Jr., Jin
Woo Jun, Dae Yong Kim, Se Chang Park (2011). Surgical removal of an anal cyst
caused by a protozoan parasite (Thelohanellus kitauei) from a koi (Cyprinus carpio). J.
Am. Vet. Med. Assoc. 238(6):784-786.
10. Sang Yoon Han, Sang Phil Shin, Ji Hyung Kim, Casiano H. Choresca Jr., Jee Eun Han,
Jin Woo Jun, Se Chang Park (2011). Prevalence and different characteristics of two
serotypes of Streptococcus parauberis isolated from the farmed olive flounder,
Paralichthys olivaceus (Temminck & Schlegel), in Korea. J. Fish Dis. 34:731-739.
11. Jee Eun Han, Casiano H. Choresca Jr., Ok Jae Koo, Hyun Ju Oh, So Gun Hong, Ji
Hyung Kim, Sang Phil Shin, Jin Woo Jun, Byeong Chun Lee, Se Chang Park (2011).
Establishment of glass catfish (Kryptopterus bicirrhis) fin-derived cells. Cell Biol. Int.
Rep. 18:e00008.
12. Sang Yoon Han, Bo Kyu Kang, Bong Jo Kang, Jong Man Kim, Jee Eun Han, Ji Hyung
Kim, Casiano Choresca Jr., Sang Phil Shin, Jin Woo Jun and Se Chang Park (2011)
123
Protective Efficacy of a combined vaccine against Edwardsiella tarda, Streptococcus
iniae, and Streptococcus parauberis in farmed olive flounder Paralichthys olivaceus.
Fish pathol. 46(4):108-111.
2010
1. Jin Woo Jun, Ji Hyung Kim, Dennis K. Gomez, Casiano H. Choresca Jr., Jee Eun
Han, Sang Phil Shin, Se Chang Park (2010). Occurrence of tetracycline-resistant
Aeromonas hydrophila infection in Korean cyprinid loach (Misgurnus
anguillicaudatus). Afr. J. Microbiol. Res. 4(9):849-855.
2. Jin-Woo Jun, Ji Hyung Kim, Casiano Choresca Jr., Dennis K. Gomez, Sang-Phil Shin,
Jee-Eun Han, Se-Chang Park (2010). Isolation of Aeromonas sobria containing
hemolysin gene from Arowana (Scleropages formosus). J. Vet. Clin. 27(1):62-65.
3. Jin-Woo Jun, Ji Hyung Kim, Jee-Eun Han, Sang-Phil Shin, Dennis K. Gomez,
Casiano Choresca Jr., Kyu-Seon Oh, Se-Chang Park (2010). Isolation of
Photobacterium damselae subsp. damselae from the giant grouper, Epinephelus
Lanceolatus. J. Vet. Clin. 27(5):618-621.
4. Jee Eun Han, Sang Phil Shin, Ji Hyung Kim, Casiano H. Choresca Jr., Jin Woo Jun,
Dennis K. Gomez, Se Chang Park (2010). Mortality of cultured koi Cyprinus carpio in
Korea caused by Bothriocephalus acheilognathi. Afr. J. Microbiol. Res. 4(7):543-546.
5. Sang-Phil Shin, Hyang Jee, Jee-Eun Han, Dennis K. Gomez, Ji Hyung Kim, Casiano
H. Choresca Jr., Jin-Woo Jun, Dae-Yong Kim, Se-Chang Park (2010). Occurrence of
goiter in flowerhorn cichlid (Family: Cichlidae) and its effect on liver. J. Vet. Clin.
27(2):202-204.
124
6. Casiano H. Choresca Jr., Dennis K. Gomez, Jee-Eun Han, Sang-Phil Shin, Ji Hyung
Kim, Jin-Woo Jun, Se-Chang Park (2010). Molecular detection of Aeromonas
hydrophila isolated from albino catfish, Clarias sp. reared in an indoor commercial
aquarium. Korean J. Vet. Res. 50(4):331-333.
125
List of conference attendance
2012
1. Jin Woo Jun, Ji Hyung Kim, Casiano H. Choresca Jr., Sang Phil Shin, Jee Eun Han,
Se Chang Park: Characterization of T4-Like Lytic Bacteriophages of Aeromonas
hydrophila pAh1-C, pAh6-C and its application for therapy. Japanese Society for Fish
Pathology conference, Japan (Shimonoseki) Sep., 2012.
2. Sang Phil Shin, Sang Yoon Han, Ji Hyung Kim, Casiano H. Choresca Jr., Jee Eun Han,
Jin Woo Jun, Se Chang Park: Molecular cloning and characterization of Cathepsin–
like protease from a scuticocliat from Philasterides dicentra-rchi. Japanese Society for
Fish Pathology conference, Japan (Shimonoseki) Sep., 2012.
3. Sang Phil Shin, Ji Hyung Kim, Casiano H. Choresca Jr., Jee Eun Han, Jin Woo Jun,
Se Chang Park: Comparision and phylogenetic analysis of Thelohanellus kitauei with
other Thelohanellus spp. Japanese Society for Fish Pathology conference, Japan
(Shimonoseki) Sep., 2012.
4. Casiano H. Choresca Jr., Su Jin Kim, Jung Taeck Kang, Ji Hyung Kim, Sang Phil Shin,
Jee Eun Han, Jin Woo Jun, Goo Jang, Byeong Chun Lee, Se Chang Park: Efficacy of
lipid based transfection in the goldfish, Carassius auratus, primary fibroblast cells.
Japan (Shimonoseki) Sep., 2012.
5. Jin Woo Jun, Rae Yeong Kim, Hong Hui Lee, Hyun Jin Lee, Se Chang Park: Control
of Aeromonas hydrophila infection in cyprinid loaches (Misgurnus anguillicaudatus)
by bacteriophage. 2012 Korean Society of Veterinary Science Conference and General
Meeting, Korea (Seoul) Oct., 2012.
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6. Ji Hyung Kim, Casiano Choresca Jr., Sang Phil Shin, Jee Eun Han, Jin Woo Jun, Do
Hyung Kang, Chul Hong Oh, Su Jin Heo, Se Chang Park: Biological control of
Aeromonas salmonicida subsp. salmonicida infection in Rainbow trout
(Oncorhynchus mykiss) using Aeromonas phage PAS-1. 2012 Korean Society of
Veterinary Science Conference and General Meeting, Korea (Seoul) Oct., 2012.
7. Sang Phil Shin, Han Sang Yoon, Ji Hyung Kim, Casiano Choresca Jr., Jee Eun Han,
Jin Woo Jun, Se Chang Park: PCR-based site-direct mutagenesis and expression of
cystein protease from a scuticociliate Philasterides dicentrarchi. 2012 Korean Society
of Veterinary Science Conference and General Meeting, Korea (Seoul) Oct., 2012.
8. Jee Eun Han, Ji Hyung Kim, Casiano Choresca Jr., Sang Phil Shin, Jin Woo Jun, Se
Chang Park: Multilocus Sequence Typing of Riemerella anatipestifer Isolates from
Ducklings with Tremor in South Korea. 2012 Korean Society of Veterinary Science
Conference and General Meeting, Korea (Seoul) Oct., 2012.
9. Jee Eun Han, Ji Hyung Kim, Casiano Choresca Jr., Sang Phil Shin, Jin Woo Jun, Se
Chang Park: Isolation of IncQ-type plasmid carrying the quinolone resistance (qnrS2)
gene from Aeromonas hydrophila. 2012 Korean Society of Veterinary Science
Conference and General Meeting, Korea (Seoul) Oct., 2012.
10. Jee Eun Han, Ji Hyung Kim, Casiano Choresca Jr., Sang Phil Shin, Jin Woo Jun, Se
Chang Park: Two Small ColE-Type Plasmid in Aeromonas spp. Carrying Quinolone
Resistance (qnrS2) Gene. 2012 Korean Society of Veterinary Science Conference and
General Meeting, Korea (Seoul) Oct., 2012.
11. Kim Mun Sup, Sang Phil Shin, Jee Eun Han, Ji Hyung Kim, Casiano Choresca Jr., Jin
Woo Jun, Se Chang Park: Antimicrobial effect of hypochlorous acid on pathogenic
microorganisms. 2012 Korean Society of Veterinary Science Conference and General
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Meeting, Korea (Seoul) Oct., 2012.
2011
1. Jin Woo Jun, Ji Hyung Kim, Casiano H. Choresca Jr., Jee Eun Han, Sang Phil Shin
and Se Chang Park: Isolation, molecular characterization and antibiotic susceptibility
of Vibrio parahaemolyticus in Korean seafood. Aquaculture Europe 2011, Greece
(Rhodes) Oct., 2011.
2. Jin Woo Jun, Ji Hyung Kim, Casiano H, Choresca Jr., Jee Eun Han, Sang Phil Shin
and Se Chang Park: Isolation and molecular detiction of Plesiomonas shigelloides
containing tetA gene from asian arowana Scleropages formosus in a Korean aquarium.
Aquaculture Europe 2011, Greece (Rhodes) Oct., 2011.
3. Jee Eun Han, Ji Hyung Kim, Casiano H. Choresca Jr., Sang Phil Shin, Jin Woo Jun,
Ji Young Chai and Se Chang Park: Quinolone resistance and their genetic
determinants in motile Aeromonas spp. from the diseased fishes and environmental
water in Korea. Aquaculture Europe 2011, Greece (Rhodes) Oct., 2011.
4. Jee Eun Han, Ji Hyung Kim, Casiano H. Choresca Jr., Sang Phil Shin, Jin Woo Jun
and Se Chang Park: Identification of tetracycline resistance gene encoded R- plasmid
in Aeromonas hydrophila from a cherry salmon. Aquaculture Europe 2011, Greece
(Rhodes) Oct., 2011.
5. Sang Phil Shin, Ji Hyung Kim, Casiano H. Choresca Jr., Jee Eun Han, Jin Woo Jun
and Se Chang Park: Rapid detection and isolation of Salmonella sp. from amphibians
and reptiles. Aquaculture Europe 2011, Greece (Rhodes) Oct., 2011.
6. Casiano H. Choresca Jr., Su Jin Kim, Jung Taeck Kang, Bego Roibas da Torre, Ji
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Hyung Kim, Sang Phil Shin, Jee Eun Han, Jin Woo Jun and Se Chang Park:
Transfection of goldfish Carassius auratus caudal fin derived primary cells.
Aquaculture Europe 2011, Greece (Rhodes) Oct., 2011.
7. Casiano H. Choresca Jr., Ji Hyung Kim, Jee Eun Han, Sang Phil Shin, Jin Woo Jun,
Byeong Chun Lee and Se Chang Park: Influence of the storage media and time on fin
explants culture in goldfish Carassius auratus. Aquaculture Europe 2011, Greece
(Rhodes) Oct., 2011.
8. Ji-Hyung Kim, Casiano H. Choresca Jr., Sang-Phil Shin, Jee-Eun Han, Jin-Woo Jun,
Se-Chang Park: Molecular identification of infectious hypodermal and Hematopoietic
Necrosis Virus (IHHNV) from Litopenaeus vannamei Shrimp Cultured in South Korea.
Aquaculture Europe 2011, Greece (Rhodes) Oct., 2011.
9. Ji-Hyung Kim, Casiano H. Choresca Jr., Sang-Phil Shin, Jee-Eun Han, Jin-Woo Jun,
Se-Chang Park: Antimicrobial resistance and clonal relatedness of Aeromonas
salmonicida isolates from cultured fish in South Korea. Aquaculture Europe 2011,
Greece (Rhodes) Oct., 2011.
10. Jin-Woo Jun, Ji-Hyung Kim, Casiano H. Choresca Jr., Sang-Phil Shin, Jee-Eun Han,
Eun-Chae Rye, Se Chang Park Vibrio parahaemolyticus in live seafood and related
environment: 2009 Korea survey. 2011 Korean Society of Veterinary Science
Conference and General Meeting, Korea (Cheonan) Oct., 2011.
11. Jin-Woo Jun, Ji-Hyung Kim, Casiano H. Choresca Jr., Jee-Eun Han, Sang-Phil Shin,
Eun-Chae Ryu, Se-Chang Park: Occurrence of Plesiomonas shigelloides infection
containing tetA gene in Asian arowana (Scleropages formosus). 2011 Korean Society
of Veterinary Science Conference and General Meeting, Korea (Cheonan) Oct., 2011.
12. Ji-Hyung Kim, Casiano H. Choresca Jr., Sang-Phil Shin, Jee-Eun Han, Jin-Woo Jun,
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Sang-Yoon Han, Do-Hyung Kang, Se-Chang Park: First detection and genome
sequencing of infectious hypodermal and hematopoietic necrosis virus (IHHNV) from
Litopenaeus vannamei shrimp cultured in South Korea. 2011 Korean Society of
Veterinary Science Conference and General Meeting, Korea (Cheonan) Oct., 2011.
13. Jee Eun Han, Ji Hyung Kim, Casiano Choresca Jr., Sang Phil Shin, Jin Woo Jun, Ji
Young Chai, Sang Yoon Han, Eun Chae Ryu, Se Chang Park: Detection of new qnrS
gene in motile Aeromonas spp. from diseased fish and water. 2011 Korean Society of
Veterinary Science Conference and General Meeting, Korea (Cheonan) Oct., 2011.
14. Casiano Choresca Jr, Ji-Hyung Kim, Jee Eun Han, Sang Phil Shin, Jin Woo Jun, Eun
Chae Ryu, Byeong Chun Lee, Se Chang Park: Culture of goldfish caudal fin explants:
Influence of storage media, time and glycerol cryopreservation. 2011 Korean Society
of Veterinary Science Conference and General Meeting, Korea (Cheonan) Oct., 2011.
15. Sang-Phil Shin, Ji-Hyung Kim, Casiano H. Chroresca Jr., Jee-Eun Han, Jin-Woo Jun,
Eun-Chae Ryu, Se-Chang Park: Phylogenetic characterization of Thelohanellus kitauei
about host specificity and tissue tropism. 2011 Korean Society of Veterinary Science
Conference and General Meeting, Korea (Cheonan) Oct., 2011.
16. Sang-Phil Shin, Ji-Hyung Kim, Casiano H. Chroresca Jr., Jee-Eun Han, Jin-Woo Jun,
Eun-Chae Ryu, Se-Chang Park: Comparison of detection methods of Salmonella sp.
from amphibians and reptiles. 2011 Korean Society of Veterinary Science Conference
and General Meeting, Korea (Cheonan) Oct., 2011.
17. Jee Eun Han, Ji Hyung Kim, Casiano Choresca Jr., Sang Phil Shin, Jin Woo Jun, Eun
Chae Ryu, Se Chang Park: Prevalence of tet gene in Aeromonas species isolated from
environmental water and cultured fish in South Korea. 2011 Korean Society of
Veterinary Science Conference and General Meeting, Korea (Cheonan) Oct., 2011.
130
18. Ji-Hyung Kim, Ji-Soo Son, Casiano-Hermopia Choresca Jr., Sang-Phil Shin, Jee-Eun
Han, Jin-Woo Jun, Do-Hyung Kang, Se-Chang Park: Isolation of a novel virulent
Myoviridae bacteriophage PAS-1 infecting Aeromonas salmoncida subsp. salmonicida.
2011 Korean Society of Veterinary Science Confer-ence and General Meeting, Korea
(Cheonan) Oct., 2011.
19. Casiano Choresca Jr., Su Jin Kim, Jung Taek Kang, Bego Roibas da Torre, Ji Hyung
Kim, Jee Eun Han, Sang Phil Shin, Jin Woo Jun, Eun Chae Ryu, Goo Jang, Byeong
Chun Lee, Se Chang Park: Transient transfection of red fluorescent protein gene in
goldfish caudal fin derived primary cells. 2011 Korean Society of Veterinary Science
Conference and General Meeting, Korea (Cheonan) Oct., 2011.
2010
1. Jin Woo Jun, Ji Hyung Kim, Dennis K. Gomez, Casiano H. Choresca Jr., Jee Eun
Han, Sang Phil Shin and Se Chang Park: Occurrence of tetracycline-resistant
Aeromonas hydrophila infection in Korean cyprinid loach Misgurnus anguillicaudatus.
Aquaculture Europe 2010, Portugal (Porto) Oct., 2010.
2. Jin Woo Jun, Ji Hyung Kim, Dennis K. Gomez, Casiano H, Choresca Jr., Jee Eun
Han, Sang Phil Shin and Se Chang Park: Isolation of Photobacterium damselae subsp.
damselae from giant grouper Epinephelus lanceolatus. Aquaculture Europe 2010,
Portugal (Porto) Oct., 2010.
3. Sang Phil Shin, Dennis K. Gomez, Ji Hyung Kim, Casiano H. Choresca Jr., Jee Eun
Han, Jin Woo Jun and Se Chang Park: Detection and genetic analysis of aqua-
birnaviruses in subclinically infected aquarium fish. Aquaculture Europe 2010,
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Portugal (Porto) Oct., 2010.
4. Sang Phil Shin, Hyang Jee, Dennis K. Gomez, Ji Hyung Kim, Casiano H. Choresca Jr.,
Jee Eun Han, Jin Woo Jun, Dae Yong Kim and Se Chang Park: Occurrence of goiter
in flowerhorn cichlid and its effect on liver. Aquaculture Europe 2010, Portugal
(Porto) Oct., 2010.
5. Casiano H. Choresca Jr., Jee Eun Han, Dennis K. Gomez, Sang Phil Shin, Ji Hyung
Kim, Jin Woo Jun and Se Chang Park: Mortality of albino catfish Clarias batrachus
caused by Aeromonas hydrophila exhibited in an indoor commercial aquarium.
Aquaculture Europe 2010, Portugal (Porto) Oct., 2010.
6. Jee Eun Han, Sang Phil Shin, Ji Hyung Kim, Dennis K. Gomez, Casiano H. Choresca
Jr., Jin Woo Jun and Se Chang Park: Mortality of cultured koi Cyprinus carpio in
Korea caused by Bothriocephalus acheilognathi. Aquaculture Europe 2010, Portugal
(Porto) Oct., 2010.
7. Jee Eun Han, Ji Hyung Kim, Dennis K. Gomez, Casiano H. Choresca Jr., Sang Phil
Shin, Jin Woo Jun and Se Chang Park: Antimicrobial resistance and its genetic
determinants in Aeromonas hydrophila from aquarium-cultured cherry salmon
Oncorhynchus masou masou. Aquaculture Europe 2010, Portugal (Porto) Oct., 2010.
8. Casiano H. Choresca Jr., Dennis K. Gomez, Ji Hyung Kim, Jee Eun Han, Sang Phil
Shin, Jin Woo Jun, Byeong Chun Lee and Se Chang Park: Cryo-banking of gold fish
fin explants using glycerol as a cryoprotectant. Aquaculture Europe 2010, Portugal
(Porto) Oct., 2010.
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2009
1. Jin Woo Jun, Ji Hyung Kim, Dennis K. Gomez, Casiano H, Choresca Jr., Jee Eun
Han, Sang Phil Shin and Se Chang Park: Mass mortality of cyprinid loach Misgurnus
anguillicaudatus caused by Aeromonas hydrophila. The 2nd FASAVA Congress 2009,
Thailand (Bangkok) Nov., 2009.
2. Jin Woo Jun, Ji Hyung Kim, Dennis K. Gomez, Casiano H, Choresca Jr., Jee Eun
Han, Sang Phil Shin and Se Chang Park: Isolation of Aeromonas sobria encoding
hemolysin gene from dragon fish Scleropages formosus. The 2nd FASAVA Congress
2009, Thailand (Bangkok) Nov., 2009.
3. Dennis K. Gomez, Casiano H, Choresca Jr., Ji Hyung Kim, Sang Phil Shin, Jee Eun
Han, Jin Woo Jun and Se Chang Park: Mortality of banded hound shark Triakis
Scyllium caused by Citrobacter koseri in a commercial aquarium. The 2nd FASAVA
Congress 2009, Thailand (Bangkok) Nov., 2009.
4. Dennis K. Gomez, Casiano H, Choresca Jr., Ji Hyung Kim, Sang Phil Shin, Jee Eun
Han, Jin Woo Jun and Se Chang Park: Molecular detection of betanodaviruses from
wild marine or freshwater fishes and invertebrates in Korea. The 2nd FASAVA
Congress 2009, Thailand (Bangkok) Nov., 2009.
5. Ji Hyung Kim, Dennis K. Gomez, Casiano H, Choresca Jr., Jee Eun Han, Jin Woo
Jun, Sang Phil Shin and Se Chang Park: Citrobacter freundii infection of doctor fish
Garra rufa obtusa with mass mortality. The 2nd FASAVA Congress 2009, Thailand
(Bangkok) Nov., 2009.
6. Ji Hyung Kim, Dennis K. Gomez, Casiano H, Choresca Jr., Jee Eun Han, Jin Woo
Jun, Sang Phil Shin and Se Chang Park: Experimental infection of aquatic animals
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with low pathogenic avian influenza virus (H9N2) of Korean isolate. The 2nd
FASAVA Congress 2009, Thailand (Bangkok) Nov., 2009.
7. Casiano H, Choresca Jr., Dennis K. Gomez, Ji Hyung Kim, Sang Phil Shin, Jee Eun
Han, Jin Woo Jun and Se Chang Park: Isolation of Edwardsiella tarda from pirarucu
Arapaima Gigas maintained in a private commercial aquarium. The 2nd FASAVA
Congress 2009, Thailand (Bangkok) Nov., 2009.
8. Casiano H, Choresca Jr., Dennis K. Gomez, Ji Hyung Kim, Sang Phil Shin, Jee Eun
Han, Jin Woo Jun and Se Chang Park: Culture of goldfish caudal fin explants: Effect
of storage time, storing media and glycerol cryopreservation. The 2nd FASAVA
Congress 2009, Thailand (Bangkok) Nov., 2009.
9. Jee Eun Han, Ji Hyung Kim, Dennis K. Gomez, Casiano H, Choresca Jr., Jin Woo
Jun, Sang Phil Shin and Se Chang Park: Isolation of a zoonotic pathogen Kluyvera
ascorbata from Egyptian fruit-bat Rousettus aegyptiacus. The 2nd FASAVA Congress
2009, Thailand (Bangkok) Nov., 2009.
10. Jee Eun Han, Ji Hyung Kim, Dennis K. Gomez, Casiano H, Choresca Jr., Jin Woo
Jun, Sang Phil Shin and Se Chang Park: Development of fish somatic cell line derived
from fin of glass catfish Kryptopterus bicirrhis. The 2nd FASAVA Congress 2009,
Thailand (Bangkok) Nov., 2009.
11. Sang Phil Shin, Dennis K. Gomez, Casiano H, Choresca Jr., Ji Hyung Kim, Jee Eun
Han, Jin Woo Jun and Se Chang Park: Morphological and molecular identification of
scuticociliate Philasterides dicentrarchi in Indo-Pacific deahorse Hippocampus kuda.
The 2nd FASAVA Congress 2009, Thailand (Bangkok) Nov., 2009.
12. Sang Phil Shin, Dennis K. Gomez, Casiano H, Choresca Jr., Ji Hyung Kim, Jee Eun
Han, Jin Woo Jun and Se Chang Park: Surgical removal of anal cyst from koi
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Cyprinus carpio koi caused by Thelohanellus kitauei. The 2nd FASAVA Congress
2009, Thailand (Bangkok) Nov., 2009.
13. Sang Phil Shin, Hyang Jee, Jee Eun Han, Dennis K. Gomez, Casiano H, Choresca Jr.,
Ji Hyung Kim, Jin Woo Jun, Dae Yong Kim and Se Chang Park: Morphological and
molecular identification of Thelohanellus kitauei caused anal cyst from Koi Cyprinus
carpio koi. Annual Meeting and International Symposium of Korean Society of
Toxicologic Pathology 2009, Korea (Seoul) Sep., 2009.
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Acknowledgements
Word of thanks is not enough to express my sincerest appreciation to all those who have
untiringly helped and assisted me in pursuing my graduate studies which was once a dream
but now reaped into reality and the rest is history so to speak both in my academic and
personal pursuits.
I wish to express my sincere gratitude to my professor/supervisor Prof. Se Chang Park
for my encouragement, creative advice and guidance during my master and doctoral
courses. His abilities to guide unique and creative ideas along with his vast technical
experience for inspection have been of enormous impression for me. I would like to thank
for the unconditional support he had extended in carrying out my experiments. The
knowledge he imparted to me is such a legacy in molding my academic endeavor. He
introduced me to the wonders of science and biotechnology. He showed me his utmost
concern not only in my academic pursuit but also in my personal life and has imparted and
encouraged the positive outlook of life among his students. This made life meaningful and
worth emulating.
My thanks should be extended to the committee members of this doctoral thesis chaired
by Prof. Byeong Chun Lee, who has encouraged me to do my best during my master and
doctoral cources. To Dr. Seong Joon Joh, Prof. Gee Wook Shin, and Prof. Mahanama de
Zoysa for their precious time and valuable knowledge shared to improve my thesis, this
deserved my utmost appreciation.
I express deepest thanks to the members of Laboratory of Aquatic Biomedicine, College
of Veterinary Medicine in SNU, Ji Hyung Kim, Sang Phil Shin, and Hyoun Joong Kim for
providing the support extended in carrying out many experiments. I wish to thank all of my
136
friends, members of College of Veterinary Medicine in SNU including epidemiology,
microbiology, avian disease, veterinary public health, etc. and I really wish to gratitude to
my sincere junior Mr. Tae-Hoon Shin in veterinary public health Lab who has helped my
experiments for many years. Also, I express deepest thanks to the staff of College of
Veterinary Medicine, Kon-Kuk University for molding me into what I am now.
My doctoral courses and study would never be completed without the support from my
family. I wish to give the biggest and greatest thanks to my mother, father, sister, and
brother. I really would like to share this moment with my wife, Ms. Jin Sun Kim who had
supported me as my lover and fiancé for more than ten years, and is still supporting me as
my wife. I am extremely thankful for her steadfast encouragement and everlasting love for
me.
Aso, I wish to express my thanks to Dr. Pantelis Katharios, Mr. Panos Kalatzis, Miss
Maria Smyrli, Mrs. Dida Kokkari, and all the other staffs in the Institute of Marine Biology,
Biotechnology and Aquaculture of Hellenic Centre for Marine Research (HCMR), Crete in
Greece for their helps and advices. Also, I’m really obliged to my friend, Prof. Roberto
Bastías in the department of Biology, Ponticia Universidad Católica de Valparaiso for his
encouragement, and I wish to cherish precious merories that we have worked together,
while drinking Raki in the downtown of Heraklion. And still, there are far too many people
to be thanked, and my thanks and gratitude are too numerous to list completely. I wish to
express lively sense of gratitude to everyone to be thanked.
All my study were financially supported by the Brain Korea 21 Program for Veterinary
Science in SNU, by a Korean Research Foundation Grant and by the Basic Science
Research Program through National Research Foundation of Korea funded by the Ministry
of Education, Science, and Technology.
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November 22, 2013
Jin Woo Jun